Stabilization of biomimetic membranes

ABSTRACT

The present disclosure provides methods, compositions, kits and apparatuses for stabilizing membranes, membrane proteins, and/or membranes containing membrane proteins using hydrophobin.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application61/716,351 filed Oct. 19, 2012, which is hereby incorporated byreference in its entirety.

BACKGROUND

Biological membrane proteins have a large variety of functions,including acting as pumps, channels, valves, energy transducers, andmechanical, thermal, and electrical sensors, among many others. Membraneproteins play a role in many important cellular activities includingenergy conversion, cell signaling, cell-cell interactions, celladhesion, cell migration, protein trafficking, viral fusion, neuralsynaptic activities and ion and metabolite transport. Membrane proteinsare embedded in the lipid bilayer of the cell membrane and are comprisedof both hydrophobic and hydrophilic moieties. Membranes comprising anartificial lipid bilayer with incorporated functional membrane proteins,such as ion channel peptides and transmembrane proteins are useful in adiverse range of technical applications. Since these proteins arenanometers in size and highly efficient, they are highly attractive foruse in artificial devices. However, their natural lipid membraneenvironment suffers from shortcomings such as low strength, necessity ofan aqueous environment, and susceptibility to chemical or bacterialdegradation. Another common problem for such membranes is the need forstability of the membranes over time and against mechanical, electricaland chemical impacts.

Because membrane proteins possess both hydrophobic and hydrophilicregions, they are difficult to solubilize, extract and purify. One ofthe challenges posed by membrane proteins is that they are subject torapid denaturation and/or aggregation in solution. Despite theavailability of a wide range of surfactants, few provide increasedand/or prolonged stability of membrane proteins in solution and/or inthe membranes. Therefore, there remains a need in the art forsurfactants capable of increasing the stability of membrane proteins.The present disclosure provides methods, compositions, kits andapparatuses to address these problems.

SUMMARY OF THE INVENTION

The present invention provides methods, compositions, kits andapparatuses for stabilizing membranes and membrane proteins usinghydrophobin.

In some embodiments, the invention provides methods of stabilizing abiomimetic membrane by adding one or more hydrophobins to the biomimeticmembrane. In some embodiments, the invention provides compositionscomprising a biomimetic membrane and one or more hydrophobins. In someembodiments, the biomimetic membrane further comprises a membraneprotein.

In some embodiments, the hydrophobin is a hydrophobin having the generalformula (I):(Y1)n-B1-(X1)a-B2-(X2)b-B3-(X3)c-B4-(X4)d-B5-(X5)e-B6-(X6)f-B7-(X7)g-B8-(Y2)m  (I)where:m and n are independently 0 to 2000; B1, B2, B3, B4, B5, B6, B7 and B8are each independently amino acids selected from Cys, Leu, Ala, Pro,Ser, Thr, Met or Gly, at least 6 of the residues B1 through B8 beingCys; X1, X2, X3, X4, X5, X6, X7, Y1 and Y2 independently represent anyamino acid; a is 1 to 50; b is 0 to 5; c is 1 to 100; d is 1 to 100; eis 1 to 50; f is 0 to 5; and g is 1 to 100.

In some embodiments, the biomimetic membrane further comprises amembrane protein. In some embodiments, the membrane protein is anaquaporin. In some embodiments, the aquaporin is AQP1. In someembodiments, the aquaporin is of plant origin. In some embodiments, theaquaporin is selected from the group consisting of a Tonoplast IntrinsicProtein, a Plasma Membrane Intrinsic Protein, and a Nodulin-26 likeIntrinsic Protein aquaporin, and mixtures and hybrids thereof. In someembodiments, the aquaporin is an aquaglyceroporin (GLpF). In someembodiments, the GLpF is selected from the group consisting of a GLPAchannel, a GLPB1 channel, a GLPB2 channel, a GLPB3 channel, and a GLPY2channel, and mixtures and hybrids thereof. In some embodiments, theaquaporin water channel is aquaporin Z (AqpZ), which is derived from E.Coli. In some embodiments, the aquaporin comprises a modified sequence.

In some embodiments, the hydrophobin has a sequence of between 40 and120 amino acids in the hydrophobin core. In some embodiments, thehydrophobin has the general formula (II):(Y1)n-B1-(X1)a-B2-(X2)b-B3-(X3)c-B4-(X4)d-B5-(X5)e-B6-(X6)f-B7-(X7)_(g)-B8-(Y2)m  (II)where:m and n are independently 0 to 20; B1, B2, B3, B4, B5, B6, B7 and B8 areeach independently amino acids selected from Cys, Leu, Ala, Pro, Ser,Thr, Met or Gly, at least 7 of the residues B1 through B8 being Cys; ais 3 to 25; b is 0 to 2; c is 5 to 50; d is 2 to 35; e is 2 to 15; f is0 to 2; and g is 3 to 35.

In some embodiments, the hydrophobin has the general formula (III):(Y1)n-B1-(X1)a-B2-B3-(X3)c-B4-(X4)d-B5-(X5)e-B6-B7-(X7)g-B8-(Y2)m  (III)where:m and n are independently 0 to 20; B1, B2, B3, B4, B5, B6, B7 and B8 areeach independently amino acids selected from Cys, Leu, Ala, Pro, Ser,Thr, Met or Gly, at least 7 of the residues B1 through B8 being Cys; ais 5 to 15; c is 5 to 40; d is 4 to 23; e is 5 to 12; and g is 6 to 21.

In some embodiments, all 8 of the residues B1 through B8 are Cys.

In some embodiments, the hydrophobin is a hydrophobin fusion protein.

In some embodiments, the hydrophobin is obtained or obtainable from afilamentous fungus. In some embodiments, the hydrophobin is obtained orobtainable from a fungus of genus selected from the group consisting ofCladosporium, Ophistoma, Cryphonectria, Trichoderma, Gibberella,Neurospora, Maganaporthe, Hypocrea, Xanthoria, Emericella, Aspergillus,Paracoccioides, Metarhizium, Pleurotus, Coprinus, Dicotyonema,Flammulina, Schizophyllum, Agaricus, Pisolithus, Tricholoma, Pholioka,Talaromyces and Agrocybe.

In some embodiments, the hydrophobin is generated in situ in thecomposition.

In some embodiments, the hydrophobin causes the equilibrium surfacetension at a water/air interface to reduce to below 70 mN/m, below 50mN/m or below 40 mN/m. In some embodiments, the hydrophobin causes thesurface shear elasticity at a water/air interface to increase to 0.3-0.6N/m or higher.

In some embodiments, the hydrophobin causes at least 1.2 fold increasein stability of the biomimetic membrane compared to the stability in theabsence of the hydrophobin. In some embodiments, the hydrophobinprovides a decrease in an equilibrium surface tension at the biomimeticmembrane below 50 mN/m. In some embodiments, the hydrophobin causes thesurface shear elasticity at the biomimetic membrane to increase to0.3-0.6 N/m or higher.

In some embodiments, the hydrophobin is a Class II hydrophobin. In someembodiments, the hydrophobin is a Class II hydrophobin having thegeneral formula (IV):(Y1)n-B1-(X1)a-B2-B3-(X3)c-B4-(X4)d-B5-(X5)e-B6-B7-(X7)g-B8-(Y2)m  (IV)where:m and n are independently 0 to 200; B1, B2, B3, B4, B5, B6, B7 and B8are each independently amino acids selected from Cys, Leu, Ala, Ser,Thr, Met or Gly, at least 6 of the residues B1 through B8 being Cys; ais 6 to 12; c is 8 to 16; d is 2 to 20; e is 4 to 12; and g is 5 to 15.

In some embodiments, the hydrophobin is a Class II hydrophobin havingthe general formula (V):(Y1)n-B1-(X1)a-B2-B3-(X3)c-B4-(X4)d-B5-(X5)e-B6-B7-(X7)g-B8-(Y2)m  (V)where:m and n are independently 0 to 10; B1, B2, B3, B4, B5, B6, B7 and B8 areeach independently amino acids selected from Cys, Leu or Ser, at least 7of the residues B1 through B8 being Cys; a is 7 to 11; c is 11; d is 4to 18; e is 6 to 10; and g is 7 to 10.

In some embodiments, all 8 of the residues B1 through B8 are Cys. Insome embodiments, the group (X3)c comprises the sequence motif ZZXZ,where Z is an aliphatic amino acid; and X is any amino acid.

In some embodiments, the hydrophobin is present in a concentration of0.1 μM-50 mM. In some embodiments, the hydrophobin is present in aconcentration of 0.1-20% by weight of the total weight of thecomposition.

In some embodiments, the hydrophobin is selected from the groupsconsisting of HFBII (SEQ ID NO: 2), HFBI (SEQ ID NO: 4), SC3 (SEQ ID NO:6), EAS (SEQ ID NO: 8) and TT1 (SEQ ID NO: 10), or a protein having atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, or at least 99% sequence identity in thehydrophobin core to any thereof. In some embodiments, the hydrophobin is“HFBII” (SEQ ID NO: 2), or a protein having at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, or atleast 99% sequence identity in the hydrophobin core thereof.

In some embodiments, the invention provides methods for preparing awater filtrate. In some embodiments, the invention provides methods forpreparing a water filtrate comprising filtering an aqueous solutionthrough a biomimetic membrane comprising hydrophobin. In someembodiments, the invention provides methods for preparing a waterfiltrate comprising filtering an aqueous solution through a membranecomprising a sandwich construction having at least two permeable supportlayers separated by at least one lipid bilayer comprising functionalaquaporin water channels and one or more hydrophobins.

In some embodiments, the invention provides methods for preparing awater filtrate comprising filtering an aqueous solution through amembrane comprising one or more vesicles (e.g. lipid and/or polymervesicles) comprising functional aquaporin water channels and one or morehydrophobins. In some embodiments, the one or more vesicles aresupported in a substrate (e.g. a microporous substrate). In someembodiments, the membrane comprises one or more vesicles (e.g. lipidand/or polymer vesicles) comprising functional aquaporin water channelsand one or more hydrophobins, wherein the vesicles are incorporated intoa thin film layer (e.g. amine functional layer) and are supported in amicroporous substrate.

In some embodiments, the invention provides membranes capable offiltering water, comprising aquaporin water transport proteins and oneor more hydrophobins.

In some embodiments, the invention provides methods of preparing a purewater filtrate, comprising filtering an aqueous solution through amembrane comprising aquaporin water transport proteins and one or morehydrophobins.

In some embodiments, the invention provides methods for the productionof salinity power using pressure retarded osmosis, the method comprisingutilizing a biomimetic membrane comprising one or more hydrophobins andaquaporin water channels to increase hydrostatic pressure, and using theincrease in hydrostatic pressure as a source of salinity power.

BRIEF DESCRIPTION OF THE DRAWINGS

The features of the invention are set forth with particularity below. Abetter understanding of the features and advantages of the presentinvention will be obtained by reference to the following detaileddescription that sets forth illustrative embodiments, in which theprinciples of the invention are utilized, and the accompanying drawingsof which:

FIG. 1 depicts a graph showing surface tension vs. time of differenttested samples.

FIG. 2 shows a KRÜS Tensiometer K100.

FIG. 3 depicts a graph showing surface tension vs. time of SDS andSDS-HFBII samples. The arrows show when the samples were dropped on thesurface.

FIG. 4 depicts a graph showing surface tension vs. time of Lipids SUVand Lipids-HFBII SUV. The arrows show when the samples were dropped onthe surface.

FIGS. 5-14 show SEQ IDs 1-10, respectively.

FIG. 15A depicts a graph showing the volume vs particle sizedistribution of solutions containing 1000 ppm of AqpZ and HBFII atconcentrations of 1000 ppm (Sample 1), 100 ppm (Sample 3) and 0 (Control1).

FIG. 15B depicts a graph showing the volume vs particle sizedistribution of solutions containing 100 ppm of AqpZ and HBFII atconcentrations of 1000 ppm (Sample 2), 100 ppm (Sample 4) and 0 (Control2).

FIG. 16 depicts a graph summarizing the water contact angle results forglass substrate, BCP coated glass, BCP coated glass after HFBmodification, and a control sample of BCP coated glass soaked in water.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure provides methods, compositions, kits andapparatuses for stabilizing a membrane protein, a membrane (e.g. abiomimetic membrane), and/or a membrane comprising a membrane proteinusing one or more hydrophobins. In some embodiments, the presentinvention provides methods, compositions, kits and apparatuses forstabilizing a biomimetic membrane using one or more hydrophobins. Insome embodiments, the present invention provides methods, compositions,kits and apparatuses for stabilizing membrane protein using one or morehydrophobins. In some embodiments, the present invention providesmethods, compositions, kits and apparatuses for stabilizing a biomimeticmembrane containing a membrane protein using one or more hydrophobins.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. All references citedherein are all incorporated by reference herein in their entireties.

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages maymean±1%.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of thisdisclosure, suitable methods and materials are described below.

Hydrophobins

In this specification the term “hydrophobin” is defined as meaning apolypeptide capable of self-assembly at a hydrophilic/hydrophobicinterface, and having the general formula (I):(Y₁)_(n)-B₁-(X₁)_(a)-B₂-(X₂)_(b)-B₃-(X₃)_(c)-B₄-(X₄)_(d)-B₅-(X₅)_(e)-B₆-(X₆)_(f)-B₇-(X₇)₈-B₈-(Y₂)_(m)  (I)wherein: m and n are independently 0 to 2000; B₁, B₂, B₃, B₄, B₅, B₆, B₇and B₈ are each independently amino acids selected from Cys, Leu, Ala,Pro, Ser, Thr, Met or Gly, at least 6 of the residues B₁ through B₈being Cys; X₁, X₂, X₃, X₄, X₅, X₆, X₇, Y₁ and Y₂ independently representany amino acid; a is 1 to 50; b is 0 to 5; c is 1 to 100; d is 1 to 100;e is 1 to 50; f is 0 to 5; and g is 1 to 100.

In some embodiments, the hydrophobin has a sequence of between 40 and120 amino acids in the hydrophobin core. In some embodiments, thehydrophobin has a sequence of between 45 and 100 amino acids in thehydrophobin core. In some embodiments, the hydrophobin has a sequence ofbetween 50 and 90, preferably 50 to 75, or 55 to 65 amino acids in thehydrophobin core. The term “the hydrophobin core” means the sequencebeginning with the residue B₁ and terminating with the residue B₈.

In the formula (I), at least 6, or at least 7, or all 8 of the residuesB₁ through B₈ are Cys.

In the formula (I), in some embodiments m is suitably 0 to 500, or 0 to200, or 0 to 100, or 0 to 20, or 0 to 10, or 0 to 5, or 0.

In the formula (I), in some embodiments n is suitably 0 to 500, or 0 to200, or 0 to 100, or 0 to 20, or 0 to 10, or 0 to 3.

In the formula (I), in some embodiments, a is 3 to 25, or 5 to 15. Inone embodiment, a is 5 to 9.

In the formula (I), in some embodiments, b is 0 to 2, or preferably 0.

In the formula (I), in some embodiments, c is 5 to 50, or 5 to 40. Insome embodiments, c is 11 to 39.

In the formula (I), in some embodiments, d is 2 to 35, or 4 to 23. Insome embodiments, d is 8 to 23.

In the formula (I), in some embodiments, e is 2 to 15, or 5 to 12. Insome embodiments, e is 5 to 9.

In the formula (I), in some embodiments, f is 0 to 2, or 0.

In the formula (I), in some embodiments, g is 3 to 35, or 6 to 21. Inone embodiment, g is 6 to 18.

In some embodiments, the hydrophobins used in the present invention havethe general formula (II):(Y₁)_(n)-B₁-(X₁)_(a)-B₂-(X₂)_(b)-B₃-(X₃)_(c)B₄-(X₄)_(d)-B₅-(X₅)_(e)-B₆-(X₆)_(f)-B₇-(X₇)₈-B₈-(Y₂)_(m)  (II)wherein: m and n are independently 0 to 20; B₁, B₂, B₃, B₄, B₅, B₆, B₇and B₈ are each independently amino acids selected from Cys, Leu, Ala,Pro, Ser, Thr, Met or Gly, at least 7 of the residues B₁ through B₈being Cys; a is 3 to 25; b is 0 to 2; c is 5 to 50; d is 2 to 35; e is 2to 15; f is 0 to 2; and g is 3 to 35.

In the formula (II), at least 7, or all 8 of the residues B₁ through B₈are Cys.

In some embodiments, the hydrophobins used in the present invention havethe general formula (III):(Y₁)_(n)-B₁-(X₁)_(a)-B₂-B₃-(X₃)c-B₄-(X₄)_(d)-B₅-(X₅)_(e)-B₆-B₇-(X₇)₈-B₈-(Y₂)_(m)  (III)wherein: m and n are independently 0 to 20; B₁, B₂, B₃, B₄, B₅, B₆, B₇and B₈ are each independently amino acids selected from Cys, Leu, Ala,Pro, Ser, Thr, Met or Gly, at least 7 of the residues B₁ through B₈being Cys; a is 5 to 15; c is 5 to 40; d is 4 to 23; e is 5 to 12; and gis 6 to 21.

In the formula (III), at least 7, or 8 of the residues B₁ through B₈ areCys.

In the formulae (I), (II) and (III), when 6 or 7 of the residues B₁through B₈ are Cys, it is preferred that the residues B₃ through B₇ areCys.

In the formulae (I), (II) and (III), when 7 of the residues B₁ throughB₈ are Cys, in some embodiments: (a) B₁ and B₃ through B₈ are Cys and B₂is other than Cys; (b) B₁ through B₇ are Cys and B₈ is other than Cys,(c) B₁ is other than Cys and B₂ through B₈ are Cys. When 7 of theresidues B₁ through B₈ are Cys, it is preferred that the other residueis Ser, Pro or Leu. In some embodiments, B₁ and B₃ through B₈ are Cysand B₂ is Ser. In some embodiments, B₁ through B₇ are Cys and B₈ is Leu.In further embodiments, B₁ is Pro and B₂ through B₈ are Cys.

The cysteine residues of the hydrophobins used in the present inventionmay be present in reduced form or form disulfide (—S—S—) bridges withone another in any possible combination. In some embodiments, when all 8of the residues B₁ through B₈ are Cys, disulfide bridges may be formedbetween one or more (preferably at least 2, more preferably at least 3,most preferably all 4) of the following pairs of cysteine residues: B₁and B₆; B₂ and B₅; B₃ and B₄; B₇ and B₈. In some embodiments, when all 8of the residues B₁ through B₈ are Cys, disulfide bridges may be formedbetween one or more (at least 2, or at least 3, or all 4) of thefollowing pairs of cysteine residues: B₁ and B₂; B₃ and B₄; B₅ and B₆;B₇ and B₈.

Examples of specific hydrophobins useful in the present inventioninclude those described and exemplified in the following publications:Linder et al., FEMS Microbiology Rev. 2005, 29, 877-896; Kubicek et al.,BMC Evolutionary Biology, 2008, 8, 4; Sunde et al., Micron, 2008, 39,773-784; Wessels, Adv. Micr. Physiol. 1997, 38, 1-45; Wösten, Annu. Rev.Microbiol. 2001, 55, 625-646; Hektor and Scholtmeijer, Curr. Opin.Biotech. 2005, 16, 434-439; Szilvay et al., Biochemistry, 2007, 46,2345-2354; Kisko et al. Langmuir, 2009, 25, 1612-1619; Blijdenstein,Soft Matter, 2010, 6, 1799-1808; Wösten et al., EMBO 1 1994, 13,5848-5854; Hakanpää et al., J. Biol. Chem., 2004, 279, 534-539; Wang etal.; Protein Sci., 2004, 13, 810-821; De Vocht et al., Biophys. J. 1998,74, 2059-2068; Askolin et al., Biomacromolecules 2006, 7, 1295-1301; Coxet al.; Langmuir, 2007, 23, 7995-8002; Linder et al., Biomacromolecules2001, 2, 511-517; Kallio et al. J. Biol. Chem., 2007, 282, 28733-28739;Scholtmeijer et al., Appl. Microbiol. Biotechnol., 2001, 56, 1-8;Lumsdon et al., Colloids & Surfaces B: Biointeifaces, 2005, 44, 172-178;Palomo et al., Biomacromolecules 2003, 4, 204-210; Kirkland and Keyhani,J. Ind. Microbiol. Biotechnol., Jul. 17 2010 (e-publication); Stübner etal., Int. J. Food Microbiol., 30 Jun. 2010 (e-publication); Laaksonen etal. Langmuir, 2009, 25, 5185-5192; Kwan et al. J. Mol. Biol. 2008, 382,708-720; Yu et al. Microbiology, 2008, 154, 1677-1685; Lahtinen et al.Protein Expr. Purif., 2008, 59, 18-24; Szilvay et al., FEBS Lett., 2007,5811, 2721-2726; Hakanpää et al., Acta Crystallogr. D. Biol.Crystallogr. 2006, 62, 356-367; Scholtmeijer et al., Appl. Environ.Microbiol., 2002, 68, 1367-1373; Yang et al, BMC Bioinformatics, 2006, 7Supp. 4, S16; WO 01/57066; WO 01/57528; WO 2006/082253; WO 2006/103225;WO 2006/103230; WO 2007/014897; WO 2007/087967; WO 2007/087968; WO2007/030966; WO 2008/019965; WO 2008/107439; WO 2008/110456; WO2008/116715; WO 2008/120310; WO 2009/050000; US 2006/0228484; and EP2042156A; the contents of which are incorporated herein by reference.

In some embodiments, the hydrophobin is a polypeptide selected from SEQID NOs: 2, 4, 6, 8 or 10, or a polypeptide having at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, or at least 99% sequence identity in the hydrophobin core to anythereof and retaining the above-described self-assembly property ofhydrophobins.

a. Sources of Hydrophobin

In one embodiment, the hydrophobin is obtained or obtainable from amicroorganism. In some embodiments, the microorganism is a bacteria or afungus, more preferably a fungus. In some embodiments, the hydrophobinis obtained or obtainable from a filamentous fungus.

In some embodiments, the hydrophobin is obtained or obtainable fromfungi of the genera Cladosporium (particularly C. fulvum or C.herbarum), Ophistoma (particularly O. ulmi), Cryphonectria (particularlyC. parasitica), Trichoderma (particularly T. harzianum, T.longibrichiatum, T. asperellum, T. Koningiopsis, T. aggressivum, T.stromaticum or T. reesei), Gibberella (particularly G. moniliformis),Neurospora (particularly N. crassa), Maganaporthe (particularly M.grisea), Hypocrea (particularly H. jecorina, H. atroviridis, H. virensor H. lixii), Xanthoria (particularly X. ectanoides and X. parietina),Emericella (particularly E. nidulans), Aspergillus (particularly A.fumigatus, A. oryzae), Paracoccioides (particularly P. brasiliensis),Metarhizium (particularly M. anisoplaie), Pleurotus (particularly P.ostreatus), Coprinus (particularly C. cinereus), Dicotyonema(particularly D. glabratum), Flammulina (particularly F. velutipes),Schizophyllum (particularly S. commune), Agaricus (particularly A.bisporus), Pisolithus (particularly P. tinctorius), Tricholoma(particularly T. terreum), Pholioka (particularly P. nameko),Talaromyces (particularly T. thermophilus) or Agrocybe (particularly A.aegerita).

b. Class I and II Hydrophobins

In the art, hydrophobins are divided into Classes I and II. It is knownin the art that hydrophobins of Classes I and II can be distinguished ona number of grounds, including solubility. As described herein,hydrophobins self-assemble at an interface (e.g., a water/air interface)into amphipathic interfacial films. The assembled amphipathic films ofClass I hydrophobins are generally re-solubilised only in strong acids(typically those having a pK_(a) of lower than 4, such as formic acid ortrifluoroacetic acid), whereas those of Class II are soluble in a widerrange of solvents.

In some embodiments, the hydrophobin is a Class II hydrophobin. In someembodiments, the hydrophobin is a Class I hydrophobin.

In some embodiments, the term “Class II hydrophobin” includes ahydrophobin (as defined and exemplified herein) having theabove-described self-assembly property at a water/air interface, theassembled amphipathic films being capable of redissolving to aconcentration of at least 0.1% (w/w) in an aqueous ethanol solution (60%v/v) at room temperature. In some embodiments, the term “Class Ihydrophobin” includes a hydrophobin (as defined and exemplified herein)having the above-described self-assembly property but which does nothave this specified redissolution property.

In some embodiments, the term “Class II hydrophobin” includes ahydrophobin (as defined and exemplified herein) having theabove-described self-assembly property at a water/air interface and theassembled amphipathic films being capable of redissolving to aconcentration of at least 0.1% (w/w) in an aqueous sodium dodecylsulphate solution (2% w/w) at room temperature. In some embodiments, theterm “Class I hydrophobin” includes a hydrophobin (as defined andexemplified herein) having the above-described self-assembly propertybut which does not have this specified redissolution property.

Hydrophobins of Classes I and II may also be distinguished by thehydrophobicity/hydrophilicity of a number of regions of the hydrophobinprotein.

In some embodiments, the term “Class II hydrophobin” includes ahydrophobin (as defined and exemplified herein) having theabove-described self-assembly property and in which the region betweenthe residues B₃ and B₄, i.e. the moiety (X₃)_(c), is predominantlyhydrophobic. In some embodiments, the term “Class I hydrophobin”includes a hydrophobin (as defined and exemplified herein) having theabove-described self-assembly property but in which the region betweenthe residues B₃ and B₄, i.e. the group (X₃)_(c), is predominantlyhydrophilic.

In some embodiments, the term “Class II hydrophobin” includes ahydrophobin (as defined and exemplified herein) having theabove-described self-assembly property and in which the region betweenthe residues B₇ and B₈, i.e. the moiety (X₇)₈, is predominantlyhydrophobic. In some embodiments, the term “Class I hydrophobin”includes a hydrophobin (as defined and exemplified herein) having theabove-described self-assembly property but in which the region betweenthe residues B₇ and B₈, i.e. the moiety (X₇)₈, is predominantlyhydrophilic.

In some embodiments, the term “Class II hydrophobin” includes ahydrophobin (as defined and exemplified herein) having theabove-described self-assembly property and in which the region betweenthe residues B₃ and B₄, i.e. the moiety (X₃)_(c), is predominantlyhydrophobic. In some embodiments, the term “Class I hydrophobin”includes a hydrophobin (as defined and exemplified herein) having theabove-described self-assembly property but in which the region betweenthe residues B₃ and B₄, i.e. the group (X₃)_(c), is predominantlyhydrophilic.

In some embodiments, the term “Class II hydrophobin” includes ahydrophobin (as defined and exemplified herein) having theabove-described self-assembly property and in which the region betweenthe residues B₇ and B₈, i.e. the moiety (X₇)₈, is predominantlyhydrophobic. In some embodiments, the term “Class I hydrophobin”includes a hydrophobin (as defined and exemplified herein) having theabove-described self-assembly property but in which the region betweenthe residues B₇ and B₈, i.e. the moiety (X₇)₈, is predominantlyhydrophilic.

The relative hydrophobicity/hydrophilicity of the various regions of thehydrophobin protein can be established by comparing the hydropathypattern of the hydrophobin using the method set out in Kyte andDoolittle, J. Mol. Biol., 1982, 157, 105-132. A computer program can beused to progressively evaluate the hydrophilicity and hydrophobicity ofa protein along its amino acid sequence. For this purpose, the methoduses a hydropathy scale (based on a number of experimental observationsderived from the literature) comparing the hydrophilic and hydrophobicproperties of each of the 20 amino acid side-chains. The program uses amoving-segment approach that continuously determines the averagehydropathy within a segment of predetermined length as it advancesthrough the sequence. The consecutive scores are plotted from the aminoto the carboxy terminus. At the same time, a midpoint line is printedthat corresponds to the grand average of the hydropathy of the aminoacid compositions found in most of the sequenced proteins. The method isfurther described for hydrophobins in Wessels, Adv. Microbial Physiol.1997, 38, 1-45.

Class II hydrophobins may also be characterized by their conservedsequences.

In one embodiment, the Class II hydrophobins used in the presentinvention have the general formula (IV):(Y₁)_(n)-B₁-(X₁)_(a)-B₂-B₃-(X₃)_(c)-B₄-(X₄)_(d)-B₅-(X₅)_(e)-B₆-B₇-(X₇)₈-B₈-(Y₂)_(m)  (IV)

wherein: m and n are independently 0 to 200; B₁, B₂, B₃, B₄, B₅, B₆, B₇and B₈ are each independently amino acids selected from Cys, Leu, Ala,Ser, Thr, Met or Gly, at least 6 of the residues B₁ through B₈ beingCys; a is 6 to 12; c is 8 to 16; d is 2 to 20; e is 4 to 12; and g is 5to 15.

In the formula (IV), in some embodiments, a is 7 to 11.

In the formula (IV), in some embodiments, c is 10 to 12. In someembodiments, c is 11.

In the formula (IV), in some embodiments, d is 4 to 18. In someembodiments, d is 4 to 16.

In the formula (IV), in some embodiments, e is 6 to 10. In someembodiments, e is 9 or 10.

In the formula (IV), in some embodiments, g is 6 to 12. In someembodiments, g is 7 to 10.

In some embodiments, the Class II hydrophobins used in the presentinvention have the general formula (V):(Y₁)_(n)-B₁-(X₁)_(a)-B₂-B₃-(X₃)_(c)-B₄-(X₄)_(d)-B₅-(X₅)_(e)-B₆-B₇-(X₇)₈-B₈-(Y₂)_(m)  (V)wherein: m and n are independently 0 to 10; B₁, B₂, B₃, B₄, B₅, B₆, B₇and B₈ are each independently amino acids selected from Cys, Leu or Ser,at least 7 of the residues B₁ through B₈ being Cys; a is 7 to 11; c is11; d is 4 to 18; e is 6 to 10; and g is 7 to 10.

In the formulae (IV) and (V), in some embodiments, at least 7 of theresidues B₁ through B₈ are Cys, or all 8 of the residues B₁ through B₈are Cys.

In the formulae (IV) and (V), in some embodiments, when 7 of theresidues B₁ through B₈ are Cys, it is preferred that the residues B₃through B₇ are Cys.

In the formulae (IV) and (V), in some embodiments, when 7 of theresidues B₁ through B₈ are Cys, it is preferred that: (a) B₁ and B₃through B₈ are Cys and B₂ is other than Cys; (b) B₁ through B₇ are Cysand B₈ is other than Cys, or (c) B₁ is other than Cys and B₂ through B₈are Cys. In some embodiments, when 7 of the residues B₁ through B₈ areCys, it is preferred that the other residue is Ser, Pro or Leu. In someembodiments, B₁ and B₃ through B₈ are Cys and B₂ is Ser. In someembodiments, B₁ through B₇ are Cys and B₈ is Leu. In some embodiments,B₁ is Pro and B₂ through B₈ are Cys.

In the formulae (IV) and (V), in some embodiments, the group (X₃)_(c)comprises the sequence motif ZZXZ, wherein Z is an aliphatic amino acid;and X is any amino acid. The term “aliphatic amino acid” means an aminoacid selected from the group consisting of glycine (G), alanine (A),leucine (L), isoleucine (I), valine (V) and proline (P).

In some embodiments, the group (X₃)_(c) comprises the sequence motifselected from the group consisting of LLXV, ILXV, ILXL, VLXL and VLXV.In some embodiments, the group (X₃)_(c) comprises the sequence motifVLXV.

In the formulae (IV) and (V), in some embodiments, the group (X₃)_(c)comprises the sequence motif ZZXZZXZ, wherein Z is an aliphatic aminoacid; and X is any amino acid. In some embodiments, the group (X₃)_(c)comprises the sequence motif VLZVZXL, wherein Z is an aliphatic aminoacid; and X is any amino acid.

In some embodiments, the hydrophobin is a polypeptide selected from SEQID NOs: 2, 4, 6, 8 or 10, or a polypeptide having at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, or at least 99% sequence identity in the hydrophobin core to anythereof. By “the hydrophobin core” is meant the sequence beginning withthe residue B₁ and terminating with the residue B₈.

In some embodiments, the hydrophobin is obtained or obtainable fromfungi of the phylum Ascomycota. In some embodiments, the hydrophobin isobtained or obtainable from fungi of the genera Cladosporium(particularly C. fulvum), Ophistoma (particularly C. ulmi),Cryphonectria (particularly C. parasitica), Trichoderma (particularly T.harzianum, T. longibrichiatum, T. asperellum, T. Koningiopsis, T.aggressivum, T. stromaticum or T. reesei), Gibberella (particularly G.moniliformis), Neurospora (particularly N. crassa), Maganaporthe(particularly M. grisea) or Hypocrea (particularly H. jecorina, H.atroviridis, H. virens or H. lixii).

In some embodiments, the hydrophobin is obtained or obtainable fromfungi of the genus Trichoderma (particularly T. harzianum, T.longibrichiatum, T. asperellum, T. Koningiopsis, T. aggressivum, T.stromaticum or T. reesei). In some embodiments, the hydrophobin isobtained or obtainable from fungi of the species T. reesei.

In some embodiments, the hydrophobin is selected from the groupconsisting of: (a) HFBII (SEQ ID NO: 2; obtainable from the fungusTrichoderma reesei); (b) HFBI (SEQ ID NO: 4; obtainable from the fungusTrichoderma reesei); (c) SC3 (SEQ ID NO: 6; obtainable from the fungusSchizophyllum commune); (d) EAS (SEQ ID NO: 8; obtainable from thefungus Neurospora crassa); and (e) TT1 (SEQ ID NO: 10; obtainable fromthe fungus Talaromyces thermophilus); or a protein having at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, or at least 99% sequence identity in the hydrophobin core toany thereof.

In some embodiments, the hydrophobin is encoded by the polynucleotideselected from the group consisting of: (a) HFBII (SEQ ID NO: 1;obtainable from the fungus Trichoderma reesei); (b) HFBI (SEQ ID NO: 3;obtainable from the fungus Trichoderma reesei); (c) SC3 (SEQ ID NO: 5;obtainable from the fungus Schizophyllum commune); (d) EAS (SEQ ID NO:7; obtainable from the fungus Neurospora crassa); and (e) TT1 (SEQ IDNO: 9; obtainable from the fungus Talaromyces thermophilus); or theprotein encoded by a polynucleotide which is degenerate as a result ofthe genetic code to the polynucleotides defined in (a) to (e) above.

In some embodiments, the hydrophobin is “HFBII” (SEQ ID NO: 2;obtainable from Trichoderma reesei) or a protein having at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, or at least 99% sequence identity in the hydrophobin corethereof.

In some embodiments, the hydrophobin may be present as an initialcomponent of the composition. In another embodiment, the hydrophobin maybe generated in situ in the composition (for example, by in situhydrolysis of a hydrophobin fusion protein).

In some embodiments, the hydrophobin may be replaced wholly or partiallywith a chaplin. Chaplins are hydrophobin-like proteins which are alsocapable of self-assembly at a hydrophobic-hydrophilic interface, and aretherefore functional equivalents to hydrophobins. Chaplins have beenidentified in filamentous fungi and bacteria such as Actinomycetes andStreptomyces. Unlike hydrophobins, they may have only two cysteineresidues and may form only one disulphide bridge. Examples of chaplinsare described in WO 01/74864, US 2010/0151525 and US 2010/0099844 and inTalbot, Curr. Biol. 2003, 13, R696-R698.

c. Assay

One property of the hydrophobins used in some embodiments of the presentinvention is the self-assembly property of the hydrophobins at ahydrophilic/hydrophobic interface.

Self-assembly can be detected by adsorbing the protein topolytetrafluoroethylene (TEFLON®) and using Circular Dichroism (CD) toestablish the change in secondary structure exemplified by theoccurrence of motifs in the CD spectrum corresponding to a newly formedα-helix) (De Vocht et al., Biophys. J. 1998, 74, 2059-2068). A fullprocedure for carrying out the CD spectral analysis can be found inAskolin et al. Biomacromolecules, 2006, 7, 1295-1301.

In some embodiments, the hydrophobins used in the present invention arecharacterized by their effect on the surface properties at an interface,e.g., at an air/water interface. The surface property may be surfacetension (especially equilibrium surface tension) or surface shearrheology, particularly the surface shear elasticity (storage modulus).

In some embodiments, the hydrophobin may cause the equilibrium surfacetension at a water/air interface to reduce to below 70 mN/m. In someembodiments, the hydrophobin may cause the equilibrium surface tensionat a water/air interface to reduce to below 50 mN/m. In someembodiments, the hydrophobin may cause the equilibrium surface tensionat a water/air interface to reduce to below 45 mN/m. In contrast, thesurface tension of pure water is 72 mN/m at room temperature. In someembodiments, such a reduction in the equilibrium surface tension at awater/air interface may be achieved using a hydrophobin concentration ofbetween 5×10⁻⁸M and 2×10⁻⁶ M, e.g., between 1×10⁻⁷M and 1×10⁻⁶ M. Insome embodiments such a reduction in the equilibrium surface tension ata water/air interface may be achieved at a temperature ranging from 0°C. to 50° C., especially room temperature. The change in equilibriumsurface tension can be measured using a tensiometer following the methoddescribed in Cox et al., Langmuir, 2007, 23, 7995-8002.

In some embodiments, the hydrophobin may cause the surface shearelasticity (G′_(s)) at a water/air interface to increase to 30-35 mN/m,40-50 mN/m, or higher. In some embodiments, the hydrophobin may causethe surface shear elasticity (G′_(s)) at a water/air interface toincrease to 0.3-0.6 N/m, or higher. In some embodiments, such a surfaceshear elasticity at a water/air interface may be achieved using ahydrophobin concentration of between 0.01-100 mM. In some embodiments,the hydrophobin is present in a concentration of 0.1 μM-50 mM. In someembodiments, the hydrophobin is present in a concentration of 0.1-20% byweight of the total weight of the composition. In some embodiments, sucha surface shear elasticity at a water/air interface may be achievedusing a hydrophobin concentration of between 0.01-50 mM. In someembodiments, such a surface shear elasticity at a water/air interfacemay be achieved using a hydrophobin concentration of between 0.01-20 mM.In some embodiments, such a surface shear elasticity at a biomimeticmembrane may be achieved at a temperature ranging from 0° C. to 50° C.,especially room temperature. The change in equilibrium surface tensioncan be measured using a rheometer following the method described in Coxet al., Langmuir, 2007, 23, 7995-8002

In some embodiments, the hydrophobin may cause the equilibrium surfacetension at a biomimetic membrane (e.g. a biomimetic membrane containingone or more membrane proteins) to reduce to below 50 mN/m. In someembodiments, such a reduction in the equilibrium surface tension at abiomimetic membrane may be achieved using a hydrophobin concentration ofbetween 0.01-100 mM, 0.01-50 mM or between 0.01-20 mM. In someembodiments, the hydrophobin is present in a concentration of 0.1 μM-50mM. In some embodiments, the hydrophobin is present in a concentrationof 0.1-20% by weight of the total weight of the composition. In someembodiments such a reduction in the equilibrium surface tension at abiomimetic membrane may be achieved at a temperature ranging from 0-90°C.

In some embodiments, the hydrophobin may cause the surface shearelasticity at a biomimetic membrane (e.g. a biomimetic membranecontaining one or more membrane proteins) to increase to 30-35 mN/m orhigher. In some embodiments, the hydrophobin may cause the surface shearelasticity at a biomimetic membrane (e.g. a biomimetic membranecontaining one or more membrane proteins) to increase to 0.3-0.6 N/m, orhigher. In some embodiments, such a surface shear elasticity at abiomimetic membrane may be achieved using a hydrophobin concentration ofbetween 0.01-100 mM, 0.01-50 mM or 0.01-20 mM. In some embodiments, thehydrophobin is present in a concentration of 0.1 μM-50 mM. In someembodiments, the hydrophobin is present in a concentration of 0.1-20% byweight of the total weight of the composition. In some embodiments, suchsurface shear elasticity at a biomimetic membrane may be achieved at atemperature ranging from 0-90° C.

In some embodiments, the hydrophobins used in the present invention arecharacterized by their effect on stabilization and/or dispersion ofmembrane proteins. In some embodiments the hydrophobins are used todisperse membrane proteins. Thus, in some embodiments, the membraneprotein molecules are held in dispersion in a medium. The inventionprovides stable dispersions of the membrane proteins. The dispersedmembrane protein can then be used in the formation of biomimeticmembranes.

In some embodiments, the hydrophobins used in the present invention arebiosurfactants. Biosurfactants are surface-active substances synthesizedby living cells. Among other properties, they have the properties ofreducing surface tension, stabilizing emulsions, promoting foaming andare generally non-toxic and biodegradable.

Examples of specific hydrophobins useful in the methods, compositions,kits and/or apparatuses of the present disclosure are listed in Table 1below.

TABLE 1 Gene, Protein NCBI accession code Organism name and versionnumber Agaricus bisporus ABH3 Y14602.1 Agaricus bisporus HYPB Y15940.1Aspergillus fumigatus HYP1/RODA L25258.1, U06121.1 Aspergillus fumigatusRODB AY057385.1 Aspergillus niger A_NIG1 XM_001394993.1 Aspergillusoryzae HYPB AB097448.1 Aspergillus oryzae ROLA AB094496.1 Aspergillusterreus A_TER XM_001213908.1 Cladosporium fulvum HCF-5 AJ133703.1Cladosporium fulvum HCF-6 AJ251294.1 Cladosporium fulvum HCF-3AJ566186.1 Cladosporium fulvum HCF-1 X98578.1 Cladosporium fulvum HCF-2AJ133700.1 Cladosporium fulvum HCF-4 AJ566187.1 Cladosporium herbarumHCH-1 AJ496190.1 Claviceps fusiformis CFTH1_I-III AJ133774.1 Clavicepsfusiformis CLF CAB61236.1 Claviceps purpurea CLP CAD10781.1 Clavicepspurpurea CPPH1_I-V AJ418045.1 Coprinus cinereus COH1 Y10627.1 Coprinuscinereus COH2 Y10628.1 Cryphonectria parasitica CRP L09559.1 Dictyonemaglabratum DGH3 AJ320546.1 Dictyonema glabratum DGH2 AJ320545.1Dictyonema glabratum DGH1 AJ320544.1 Emericella nidulans RODA M61113.1Emericella nidulans DEWA U07935.1 Flammulina velutipes FVH1 AB026720.1Flammulina velutipes FvHYD1 AB126686.1 Gibberella moniliformis HYD5, GIMAY158024.1 Gibberella moniliformis HYD4 AY155499.1 Gibberellamoniliformis HYD1 AY155496.1 Gibberella moniliformis HYD2 AY155497.1Gibberella moniliformis HYD3 AY155498.1 Gibberella zeae GIZ, FG01831.1XP_382007.1 Lentinula edodes Le.HYD1 AF217807.1 Lentinula edodes Le.HYD2AF217808.1 Magnaporthe grisea MGG4 XM_364289.1 Magnaporthe grisea MGG2XM_001522792.1 Magnaporthe grisea MHP1, MGG1 AF126872.1 Magnaporthegrisea MPG1 L20685.2 Metarhizium anisopliae SSGA M85281.1 Neurosporacrassa NCU08192.1 AABX01000408.1 Neurospora crassa EAS AAB24462.1Ophiostoma ulmi CU U00963.1 Paracoccidioides brasilensis PbHYD2AY427793.1 Paracoccidioides brasilensis PbHYD1 AF526275.1 Passalorafulva PF3 CAC27408.1 Passalora fulva PF1 CAC27407.1 Passalora fulva PF2CAB39312.1 Pholiota nameko PNH2 AB079129.1 Pholiota nameko PNH1AB079128.1 Pisolithus tinctorius HYDPt-1 U29605.1 Pisolithus tinctoriusHYDPt-2 U29606.1 Pisolithus tinctorius HYDPt-3 AF097516.1 Pleurotusostreatus POH2 Y14657.1 Pleurotus ostreatus POH3 Y16881.1 Pleurotusostreatus VMH3 AJ238148.1 Pleurotus ostreatus POH1 Y14656.1 Pleurotusostreatus FBHI AJ004883.1 Schizophyllum commune SC4 M32330.1Schizophyllum commune SC1, 1G2 X00788.1 Schizophyllum commune SC6AJ007504.1 Schizophyllum commune SC3 AAA96324.1 Talaromyces thermophilusTT1 Trichoderma harzianum QID3 X71913.1 Trichoderma harzianum SRH1Y11841.1 Trichoderma reesei HFBII P79073.1 Trichoderma reesei HFBIP52754.1 Tricholoma terreum HYD1 AY048578.1 Verticillium dahliae VEDAAY89101.1 Xanthoria ectaneoides XEH1 AJ250793.1 Xanthoria parietinaXPH1 AJ250794.1

d. Fusion Proteins

The hydrophobin of the present disclosure includes fusion proteins of ahydrophobin and another polypeptide as well as conjugates of hydrophobinand other molecules such as polysaccharides.

In some embodiments, the hydrophobin is a hydrophobin fusion protein.The term “fusion protein” includes a hydrophobin sequence (as definedand exemplified above) bonded to a further peptide sequence (describedherein as “a fusion partner”) which does not occur naturally in ahydrophobin.

In some embodiments, the fusion partner may be bonded to the aminoterminus of the hydrophobin core, thereby forming the group (Y₁)_(m). Insome embodiments, m may range from 1 to 2000, or 2 to 1000, or 5 to 500,or 10 to 200, or 20 to 100.

In some embodiments, the fusion partner may be bonded to the carboxylterminus of the hydrophobin core, thereby forming the grow p (Y₂)_(n).In some embodiments, n may range from 1 to 2000, or 2 to 1000, or 5 to500, or 10 to 200, or 20 to 100.

In some embodiments, fusion partners may be bonded to both the amino andcarboxyl termini of the hydrophobin core. In some embodiments, thefusion partners may be the same or different, and may have amino acidsequences having the number of amino acids defined above by the statedvalues of m and n.

In some embodiments, the hydrophobin is not a fusion protein and m and nare 0.

Membrane Proteins

In some embodiments, the biomimetic membranes describe herein compriseone or more membrane proteins. In some embodiments, the biomimeticmembranes further comprise one or more hydrophobin. The membrane proteinto be incorporated into a membrane according to the invention can be anymembrane protein.

In some embodiments the hydrophobins described herein are used tostabilize membrane proteins. The term stabilize includes disperse and/oremulsify. Thus, in some embodiments, the membrane protein molecules areheld in dispersion in a medium. The invention provides stabledispersions of the membrane proteins. The dispersed membrane protein canthen be used in the formation of biomimetic membranes.

Examples are G-protein coupled receptors such as odorant receptors,rhodopsin receptors, in particular bovine rhodopsin receptors, rhodopsinpheromone receptors, peptide hormone receptors, taste receptors, GABAreceptors, opiate receptors, serotonin receptors, Ca²⁺ receptor,melanopsin, neurotransmitter receptors, ligand gated, voltage gated ormechanically gated such as acetylcholine (ACh), nicotinic, adrener,norepinephrine, catecholamines, L-dopa, dopamine and serotonin—biogenicamines, endorphins/enkephalins—neuropeptide receptors, kinases such asserin/threonin kinases, cytoplasmic tyrosine kinases, receptor tyrosinekinases, phosphatases proteases, inactive kinases, porins/channels suchas chloride channels, potassium channels, sodium channels, OMP proteins,ABC transporter (ATP-Binding Cassette-Transporter) such as amino acidtransporter, Na-glucose transporter, Na⁺/iodide transporter, iontransporters such as Light Harvesting Complex, cytochrome c oxidase, ATPase Na/K, H/K, Ca, cell adhesion receptors such as metallo proteases,integrins, catherins.

In some embodiments, the biomimetic membranes comprise one or moremembrane proteins, such as channel forming molecules, e.g. certainpeptides or peptide like molecules including amphotericin B,alamethicin, valinomycin, gramicidin A and their dimers, oligomers andanalogues thereof; or transmembrane proteins, e.g. aquaporin waterchannels, Fas protein, DsbB, CFTR, alpha-haemolysin, VDAC, and OmpG.

In some embodiments, the biomimetic membranes comprise one or moretransmembrane proteins, e.g. aquaporin water channels. In someembodiments, the biomimetic membranes comprise one or more aquaporinwater channels

a. Aquaporin

Living cells are enclosed by a lipid bilayer membrane, separating thecells from other cells and their extracellular medium. Lipid bilayermembranes are essentially impermeable to water, ions, and other polarmolecules; yet, in many instances, such entities need to be rapidly andselectively transported across a membrane, often in response to anextra- or intracellular signal. The water-transporting task isaccomplished by aquaporin water channel proteins (Preston, G. M., P.Piazza-Carroll, W. B. Guggino, and P. Agre. (1992). Appearance of waterchannels in Xenopus oocytes expressing red cell CHIP28 water channel.Science, 256, 385-387). Aquaporins are crucial for life in any form andthey are found in all organisms, from bacteria via plants to man.Aquaporins facilitate rapid, highly selective water transport, thusallowing the cell to regulate its volume and internal osmotic pressureaccording to hydrostatic and/or osmotic pressure differences across thecell membrane. The physiological importance of the aquaporin in humansis perhaps most conspicuous in the kidney, where about 150-200 liters ofwater need to be reabsorbed from the primary urine each day, that is,aquaporin facilitated water transport is invoked when water rapidly mustbe retrieved from a body fluid. In kidneys, this is made possible mainlyby two aquaporins denoted AQP1 and AQP2 (11 different aquaporins areknown in humans). In plants, aquaporins are also critical for waterabsorption in the root and for maintaining the water balance throughoutthe plant (Agre, P., M. Bonhivers, and M. J. Borgnia. (1998) Theaquaporins, blueprints for cellular plumbing systems. Journal ofBiological Chemistry, 273, 14659-14662; Borgnia, M., S, Nielsen, A.Engel, and P. Agre. (1999). Cellular and molecular biology of theaquaporin water channels. Annual Review of Biochemistry, 68, 425-458).In plants, water is absorbed by the same osmotic forces as thisinvention intends to use in a PRO system in the production of salinitypower.

Studies of water transport in various organisms and tissues suggestedthat aquaporins have a narrow pore preventing any flow of largemolecules, ions (salts) and even protons (H₃O+) and hydroxyl ions (OH—)while maintaining an extremely high water permeation rate; about 10⁹molecules H₂O per channel per second (Agre, P., M. Bonhivers, and M. J.Borgnia. (1998) the aquaporins, blueprints for cellular plumbingsystems. Journal of Biological Chemistry, 273, 14659-14662, Borgnia, M.,S, Nielsen, A. Engel, and P. Agre. (1999). Cellular and molecularbiology of the aquaporin water channels. Annual Review of Biochemistry,68, 425-458). Until 2000 and 2001, where the first high-resolution 3Dstructure of AQP1 and that of the related glycerol-conducting bacterialchannel protein aquaglyceroporin GlpF were reported (Fu, D., Libson, A.,Miercke, L. J., Weitzman, C., Nollert, P., Krucinski, J., and Stroud, R.M. (2000). Structure of a glycerol-conducting channel and the basis forits selectivity, Science 290, 481-6; Murata, K., Mitsuoka, K., Hirai,T., Walz, T., Agre, P., Heymann, J. B., Engel, A., and Fujiyoshi, Y.(2000). Structural determinants of water permeation through aquaporin-1,Nature 407, 599-605), little was known about the origin of waterselectivity.

However, based on the experimental structures, detailed computer modelswere put forward explaining not only the high permeation rate and thestrict water selectivity but also the ability of aquaporins to preventproton leakage (de Groot, B. L., and Grubmuller, H. (2001). Waterpermeation across biological membranes: mechanism and dynamics ofaquaporin-1 and GlpF, Science 294, 2353-2357; de Groot, B. L., Frigato,T., Helms, V. and Grubmuller, H. (2003). The mechanism of protonexclusion in the aquaporin-1 channel, Journal of Molecular Biology 333,279-293; Tajkhorshid, E., Nollert, P., Jensen, M. O., Miercke, L. J.,O'Connell, J., Stroud, R. M., and Schulten, K. (2002). Control of theselectivity of the aquaporin water channel family by globalorientational tuning, Science 296, 525-530; Jensen, M. O., Tajkhorshid,E., and Schulten, K. (2003). Electrostatic tuning of permeation andselectivity in aquaporin water channels, Biophysical Journal 85,2884-2899; Zhu, F., Tajkhorshid, E. and Schulten, K. (2003). Theory andsimulation of water permeation in aquaporin-1. Biophysical Journal, 86,50-57; Burykin and A. Warshel (2003). What really prevents protontransport through aquaporin? Charge self-energy vs. proton wireproposals, Biophysical Journal 85, 3696-3706; Ilan, B., Tajkhorshid, E.,Schulten, K. and Voth, G. (2004). The mechanism of proton exclusion inaquaporin water channels. PROTEINS: Structure, Function, andBioinformatics, 55, 223-228; Chakrabarti, N., Tajkhorshid, E., Roux, B.and Pommes, R. (2004). Molecular basis of proton blockage in aquaporins,Structure 12, 65-74). In essence, the architecture of the aquaporinchannel allows water molecules to pass only in a single file whileelectrostatic tuning of the channel interior controls aquaporinselectivity against any charged species, that is, trans-port of any salt(ion) as well as protons and hydroxyl ions is abrogated (the highpermeation rate and the strict water selectivity but also the ability ofaquaporins to prevent proton leakage (de Groot, B. L., and Grubmuller,H. (2001). Water permeation across biological membranes: mechanism anddynamics of aquaporin-1 and GlpF, Science 294, 2353-2357; de Groot, B.L., Frigato, T., Helms, V. and Grubmuller, H. (2003). The mechanism ofproton exclusion in the aquaporin-1 channel, Journal of MolecularBiology 333, 279-293; Tajkhorshid, E., Nollert, P., Jensen, M. O.,Miercke, L. J., O'Connell, J., Stroud, R. M., and Schulten, K. (2002).Control of the selectivity of the aquaporin water channel family byglobal orientational tuning, Science 296, 525-530; Jensen, M. O.,Tajkhorshid, E., and Schulten, K. (2003). Electrostatic tuning ofpermeation and selectivity in aquaporin water channels, BiophysicalJournal 85, 2884-2899; Zhu, F., Tajkhorshid, E. and Schulten, K. (2003).Theory and simulation of water permeation in aquaporin-1. BiophysicalJournal, 86, 50-57; Burykin and A. Warshel (2003). What really preventsproton transport through aquaporin? Charge self-energy vs. proton wireproposals, Biophysical Journal 85, 3696-3706; Ilan, B., Tajkhorshid, E.,Schulten, K. and Voth, G. (2004). The mechanism of proton exclusion inaquaporin water channels PROTEINS: Structure, Function, andBioinformatics, 55, 223-228; Chakrabarti, N., Tajkhorshid, E., Roux, B.and Pommes, R. (2004). Molecular basis of proton blockage in aquaporins,Structure 12, 65-74). In short, this shows the high selectivity of theaquaporin water pore.

Each unit in an aquaporin channel transports about 10⁹ H₂Omolecules/sec, i.e., about 410⁹ molecules/channel/sec. Hence, 1 g ofaquaporin is capable of transporting about 720 liter of water/sec atvery high pressure.

The term “aquaporin family of membrane proteins” as used herein includesalso the GLpF proteins which in addition to water molecules alsochannels glycerol.

Transmembrane proteins different from aquaporins suitable for inclusionin the membranes of the present invention are for instance selectedfrom, but not limited to, any transmembrane protein found in theTransporter Classification Database (TCDB). TCDB is accessible at theTCDB website.

Examples of transmembrane proteins included in the present inventionfrom TCDB are: Aerolysin channel-forming toxin, Agrobacterialtarget-host cell-membrane anion channel, a-Hemolysin channel-formingtoxin, Alamethicin channel, Alginate export porin, Amoebapore,Amphipathic peptide mastoparan, Amyloid b-protein peptide, Animalinward-rectifier K⁺ channel, Annexin, Apoptosis regulator, ArpQ holin,AS-48, ATP-gated cation channel, Autotransporter, Bacillus subtilis j29holin, Bacterial type III-target cell pore, Bactericidalpermeability-increasing protein, Bacteriocin AS-48 cyclic polypeptide,Bacteriorhodopsin, Beticolin channel, BlyA holing, Botulinum and tetanustoxin, Brucella-Rhizobium porin, Campylobacter jejuni major outermembrane porin, Cathilicidin, cation channel, Cation-channel-formingheat-shock protein 70, Cecropin, Channel-forming Bacillus anthraxprotective antigen, Channel-forming ceramide, Channel-forming colicin,Channel-forming colicin V, Channel-forming d-endotoxin insecticidalcrystal protein, Channel-forming e-toxin, Channel-forming leukocidincytotoxin, Chlamydial porin, Chloride channel, Chloroplast membraneanion-channel-former, Chloroplast outer-membrane solute channel,Cholesterol-binding thiol-activated cytolysin, Clostridial cytotoxin,Complement protein C9, Complexed polyhydroxybutyrate-Ca²⁺ channel,Corynebacterial porin, Cph1 holin, C-type natriuretic peptide,Cyanobacterial porin, Cyclodextrin porin, Cytohemolysin, Cytotoxicamylin, Defensin, Dermaseptin, Diphtheria toxin, Divergicin A, Earthwormlysenin toxin, Envelope virus E1 channel, Epithelial chloride channel,Epithelial Na⁺ channel, FadL outer-membrane protein, Fusobacterialouter-membrane porin, Gap-junction-forming connexin,Gap-junction-forming innexin, General bacterial porin, Glucose-selectiveOprB porin, Glutamate-gated ion channel of neurotransmitter receptors,gp91^(Phox) phagocyte NADPH-oxidase-associated cyt b₅₅₈ H⁺-channel,Gramicidin A channel, H⁺- or Na⁺-translocating bacterial flagellarmotor, H⁺- or Na⁺-translocating bacterial MotAB flagellar motor/ExbBDouter-membrane transport, Helicobacter outer membrane porin, HP1 holin,Influenza virus matrix-2 channel, Insect defensin, Intracellularchloride channel, j11 holin, jAdh holing, jU53 holin, Lactacin X,Lacticin 481, Lactocin S, Lactococcin 972, Lactococcin A, Lactococcin G,Large-conductance mechanosensitive ion channel, lholin S, Ligand-gatedion channel of neurotransmitter receptors, LrgA holin, LydA holin,Magainin, Major intrinsic protein, Melittin, Metal-ion transporter(channel), Microcin E492, Mitochondrial and plastid porin, Mycobacterialporin, Nisin, Nonselective cation channel-1, Nonselective cationchannel-2, Nucleoside-specific channel-forming outer-membrane porin,OmpA-OmpF porin, OmpG porin, Organellar chloride channel,Outer-bacterial-membrane secretin, Outer-membrane auxiliary protein,Outer-membrane factor, Outer-membrane fimbrial usher porin,Outer-membrane porin, Outer-membrane receptor, P2 holin TM, P21 holin S,Pediocin, Phospholemman, Pilosulin, Plant defensin, Plant plasmodesmata,Plant thionine, Plantaricin EF, Plantaricin JK, Plastid outer-envelopeporin of 16 kDa, Plastid outer-envelope porin of 21 kDa, Plastidouter-envelope porin of 24 kDa, Polycystin cation channel, Polyglutamineion channel, Pore-forming equinatoxin, Pore-forming hemolysin E,Pore-forming RTX toxin, PRD1 holin M, Prion peptide fragment,Pseudomanas syringae HrpZ target-host cell-membrane, Pseudomonas OprPporin, Raffinose porin, Rhodobacter PorCa porin,Ryanodine-inositol-1,4,5-trisphosphate receptor Ca⁺ channel, Saponinchannel, Shiga toxin B-chain, Short-chain amide and urea porin,Small-conductance mechanosensitive ion channel, Sugar porin,Syringomycin channel, Syringopeptin channel, T4 holin, T4 Immunityholing, T7 holin, Tachyplesin, Tolaasin channel,TonB-ExbB-ExbD/TolA-TolQ-TolR of energizers for outer-membrane receptor(OMR)-medi-Transient receptor potential Ca⁺ channel, Tripartitehemolysin BL, Two-partner secretion porin, Type B influenza virus NBchannel, Urea transporter (channel), Urea/amide channel, Vacuolatingcytotoxin, Vibrio chitoporin/Neisseria porin, Voltage-gated ion channelsuperfamily, Whipworm stichosome porin, Yeast killer toxin Kl, Yeaststretch-activated cation-selective, Ca⁺ channel

In some embodiments, the aquaporin water channel is selected from thegroup consisting of aquaglyceroporins (GLpF), such as a GLPA channel, aGLPB1 channel, a GLPB2 channel, a GLPB3 channel, and a GLPY2 channel,and mixtures and hybrids thereof. In some embodiments, the aquaporinwater channel is aquaporin Z (AqpZ), which is derived from E. Coli. Insome embodiments, the aquaporin channels are modified. In someembodiments, AqpZ can be modified to fulfill a desired application thatmay be different from the protein's original function. For example, bysimply changing a particular amino acid residue near the center of thewater channel to cysteine, the Aquaporins produced would bind any freeMercury in the solution and cease transporting water due to theblockage. Thus, these mutant proteins used in a membrane device coulddetect Mercury contamination in a water sample by simply ceasing flowwhen the concentration of the toxic substance rises too high.

In some embodiments, one or more hydrophobins described herein are usedto disperse aquaporin proteins. Thus, in some embodiments, the aquaporinprotein molecules are held in dispersion in a medium. The inventionprovides stable dispersions of the aquaporin proteins. The dispersedaquaporin protein can then be used in the formation of biomimeticmembranes. In some embodiments, the biomimetic membranes furthercomprise or more hydrophobins in combination with one or moreaquaporins. In some embodiments, the hydrophobin is selected from thegroup consisting of: (a) HFBII (SEQ ID NO: 2; obtainable from the fungusTrichoderma reesei); (b) HFBI (SEQ ID NO: 4; obtainable from the fungusTrichoderma reesei); (c) SC3 (SEQ ID NO: 6; obtainable from the fungusSchizophyllum commune); (d) EAS (SEQ ID NO: 8; obtainable from thefungus Neurospora crassa); and (e) TT1 (SEQ ID NO: 10; obtainable fromthe fungus Talaromyces thermophilus); or a protein having at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, or at least 99% sequence identity in the hydrophobin core toany thereof.

In some embodiments, the hydrophobin is “HFBII” (SEQ ID NO: 2;obtainable from Trichoderma reesei) or a protein having at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, or at least 99% sequence identity in the hydrophobin corethereof.

In some embodiments, the dispersed medium comprise HFBII and one or moreaquaporin water channels selected from the group consisting ofaquaglyceroporins (GLpF), such as a GLPA channel, a GLPB1 channel, aGLPB2 channel, a GLPB3 channel, and a GLPY2 channel, and mixtures andhybrids thereof. In some embodiments, the dispersed medium comprisesHFBII and AqpZ. In some embodiments, the aquaporin channels aremodified.

In some embodiments, the biomimetic membranes comprise HFBII and one ormore aquaporin water channels selected from the group consisting ofaquaglyceroporins (GLpF), such as a GLPA channel, a GLPB1 channel, aGLPB2 channel, a GLPB3 channel, and a GLPY2 channel, and mixtures andhybrids thereof. In some embodiments, the biomimetic membrane comprisesHFBII and AqpZ. In some embodiments, the aquaporin channels aremodified.

Biomimetic Membranes

In one aspect, the present invention provides biomimetic membranes. Theterm biomimetic membrane includes one or more membranes or walls orshells. The biomimetic membranes include vesicles (e.g. liposomes,micelles, polymerosome, nanoparticles and microbubbles) surrounding aninternal void that could be empty or filled, e.g. filled with a gas,liquid or precursor thereto. The biomimetic membranes include planarbiomimetic membranes as well as solid-supported membranes such assolid-supported lipid bilayers and tethered lipid bilayers, or membranescomprising vesicles. In some embodiments, the biomimetic membranescomprise one or more lipids and/or one or more polymers. The term lipidsincludes agents exhibiting amphipathic characteristics causing it tospontaneously adopt an organized structure in water wherein thehydrophobic portion of the molecule is sequestered away from the aqueousphase. In some embodiments, the biomimetic membranes comprisepolymerizable lipids. In some embodiments, the biomimetic membranescomprise one or more lipids, at least one of which is polymerizable. Insome embodiments, the biomimetic membranes also contain one or morepolypeptides, and/or other functional molecules. The biomimeticmembranes of the invention may also include any other materials orcombination thereof known to those skilled in the art as suitable forbiomimetic membrane construction.

a. Lipids

In one aspect, the biomimetic membranes of the invention comprise one ormore lipid. Examples of useful lipids for the formation of lipidmembranes (e.g. monolayer, bilayers, planar or vesicles) to be used inthe biomimetic membranes of the invention, include but are not limitedto:

-   (i) Phosphatidylcholines: 1,2-dimyristoylphosphatidylcholine (DMPC);    1,2-dipalmitoylphosphatidylcholine (DPPC);    1,2-distearoylphosphatidylcholine (DSPC);    1,2-dioleoylphosphatidylcholine (DOPC);    1,2-dimyristoleoylphosphatidylcholine;    1,2-dipalmitoleoylphosphatidylcholine;    1,2-dipetroselinoylphosphatidylcholine;    1,2-dielaidoylphosphatidylcholine;    1,2-dilinoleoylphosphatidylcholine;    1,2-dilinolenoylphosphatidylcholine;    1,2-dieicosenoylphosphatidylcholine;    1,2-diarachidonoylphosphatidylcholine;    1,2-dierucoylphosphatidylcholine; 1,2-dnervonoylphosphatidylcholine;    1-palmitoyl-2-oleoylphosphatidylcholine (POPC);    1-palmitoyl-2-linoleoylphosphatidylcholine;    1-palmitoyl-2-arachidonoylphosphatidylcholine;    1-palmitoyl-2-docosahexaenoylphosphatidylcholine;    1-stearoyl-2-oleoylphosphatidylcholine (SOPC);    1-stearoyl-2-linoleoylphosphatidylcholine;    1-stearoyl-2-arachidonoylphosphatidylcholine;    1-stearoyl-2-docosahexaenoylphosphatidylcholine;    1-oleoyl-2-palmitoylphosphatidylcholine;    1-oleoyl-2-stearoylphosphatidylcholine;    1,2-didocosahexaenoylphosphatidylcholine;-   (ii) Phosphatidylethanolamines:    1,2-dimyristoylphosphatidylethanolamine (DMPE);    1,2-dipalmitoylphosphatidylethanolamine (DPPE);    1,2-distearoylphosphatidylethanolamine (DSPE);    1,2-dioleoylphosphatidylethanolamine (DOPE);    1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE);    1-palmitoyl-2-linoleoylphosphatidylethanolamine;    1-palmitoyl-2-arachidonoylphosphatidylethanolamine;    1-palmitoyl-2-docosahexaenoylphosphatidylethanolamine;    1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE);    1-stearoyl-2-linoleoylphosphatidylethanolamine;    1-stearoyl-2-arachidonoylphosphatidylethanolamine;    1-stearoyl-2-docosahexaenoylphosphatidylethanolamine;    1,2-dielaidoylphosphatidylethanolamine;    1,2-dilinoleoylphosphatidylethanolamine;    1,2-dilinolenoylphosphatidylethanolamine;    1,2-diarachidonoylphosphatidylethanolamine;    1,2-didocosahexaenoylphosphatidylethanolamine;    1,2-dipalmitoleoylphosphatidylethanolamine;-   (iii) Phosphatidylglycerols: 1,2-dimyristoylphosphatidylglycerol    (DMPG); 1,2-dipalmitoylphosphatidylglycerol (DPPG);    1,2-distearoylphosphatidylglycerol (DSPG);    1,2-dioleoylphosphatidylglycerol (DOPG);    1-palmitoyl-2-oleoylphosphatidylglycerol (POPG);    1-palmitoyl-2-linoleoylphosphatidylglycerol;    1-palmitoyl-2-arachidonoylphosphatidylglycerol;    1-palmitoyl-2-docosahexaenoylphosphatidylglycerol;    1-stearoyl-2-oleoylphosphatidylglycerol (SOPG);    1-stearoyl-2-linoleoylphosphatidylglycerol;    1-stearoyl-2-arachidonoylphosphatidylglycerol;    1-stearoyl-2-docosahexaenoylphosphatidylglycerol;-   (iv) Phosphatidylserines: 1-palmitoyl-2-oleoylphosphatidylserine    (POPS); 1-palmitoyl-2-linoleoylphosphatidylserine;    1-palmitoyl-2-arachidonoylphosphatidylserine;    1-palmitoyl-2-docosahexaenoylphosphatidylserine;    1-stearoyl-2-oleoylphosphatidylserine (SOPS);    1-stearoyl-2-linoleoylphosphatidylserine;    1-stearoyl-2-arachidonoylphosphatidylserine;    1-stearoyl-2-docosahexaenoylphosphatidylserine;    1,2-dimyristoylphosphatidylserine (DMPS);    1,2-dipalmitoylphosphatidylserine (DPPS);    1,2-distearoylphosphatidylserine (DSPS);    1,2-dioleoylphosphatidylserine (DOPS);    1,2-didocosahexaenoylphosphatidylserine;    1,2-dierucoylphosphatidylserine;-   (v) Special lipids: Cardiolipin; Bipolar lipids;-   (vi) Polymerizable lipids:    1,2-di-10,12-tricosadiynoyl-sn-glycero-3-phosphocholine (DTPC);    1,2-di-10,12-tricosadiynoyl-sn-glycero-3-phosphoethanolamine (DTPE);    1-palmitoyl-2,10,12-tricosadiynoyl-sn-glycero-3-phosphoethanolamine    (PTPE); (DC8,9PC    [1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine]; diPhyPC    [1,2-diphytanoyl-sn-glycero-3-phosphocholine]-   (vii) Natural lipid extracts: Egg yolk phosphatidylcholine; Bovine    heart phosphatidylcholine; Brain phosphatidylcholine; Bovine liver    phosphatidylcholine; Soybean phosphatidylcholine; E. Coli    phosphatidylethanolamine; Bovine Heart phosphatidylethanolamine;    Brain phosphatidylethanolamine; Bovine Liver    phosphatidylethanolamine; Egg phosphatidylethanolamine; Bovine liver    phosphatidylinositol; Soybean phosphatidylinositol; Brain    phosphatidylserine; Soy phosphatidylserine.

Useful lipids for reconstitution of aquaporins and/or hydrophobins andformation of lipid bilayers are: POPC, DPPC, ceramide and mixturesthereof.

In some embodiments, the biomimetic membranes of the invention compriseone or more polymerizable lipid. Examples of polymerizable lipidsinclude but are not limited to, diyne PC and diynePE, for example1,2-bis(10,12-tricosadiynoyl-sn-glycero-3-phosphocoline. In someembodiments, the biomimetic membrane of the invention comprise at least0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 70%, 80%, 90% or 100% of polymerizable lipids. In some embodiments,the biomimetic membranes of the invention comprise at least 25% ofpolymerizable lipids. In some embodiments, the biomimetic membranes ofthe invention comprise at least 50% of polymerizable lipids. In someembodiments, the polymerizable lipid may comprise a polymerizable groupattached to a lipid molecule. The biomimetic membranes may also containlipids that are not polymerizable, lipids conjugated to a functionalmoiety (such as a targeting agent), and lipids with a positive,negative, or neutral charge.

In some embodiments, the biomimetic membranes of the invention compriseone or more neutral phospholipids. Examples of neutral phospholipidsinclude, but are not limited to, hydrogenated phosphatidyl choline(HSPC), dipalmitoyl-, distearoyl- and diarachidoyl phosphatidylcholine(DPPC, DSPC, DAPC). In some embodiments, the biomimetic membranes of theinvention comprise at least 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90% or 100% of neutralphospholipids. In some embodiments, the biomimetic membranes of theinvention comprise at least 10% of neutral phospholipids. In someembodiments, the biomimetic membranes of the invention comprise at least30% of neutral phospholipids. In some embodiments, the biomimeticmembranes of the invention comprise at least 45% of neutralphospholipids.

In some embodiments, the biomimetic membranes of the invention compriseone or more negatively charged phospholipids. Examples of negativelycharged phospholipids include, but are not limited to, dipalmitoyl anddistearoyl phosphatidic acid (DPPA, DSPA), dipalmitoyl and distearoylphosphatidyls erine (DPPS, DSPS), phosphatidyl glycerols such asdipalmitoyl and distearoyl phosphatidylglycerol (DPPG, DSPG). In someembodiments, the biomimetic membranes of the invention comprise at least0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 70%, 80%, 90% or 100% of negatively charged phospholipids. In someembodiments, the biomimetic membranes of the invention comprise at least2% of negatively charged phospholipids. In some embodiments, thebiomimetic membranes of the invention comprise at least 5% of negativelycharged phospholipids. In some embodiments, the biomimetic membranes ofthe invention comprise at least 10% of negatively charged phospholipids.In some embodiments, the biomimetic membranes of the invention compriseat least 25% of negatively charged phospholipids. In some embodiments,the biomimetic membranes of the invention comprise at least 30% ofnegatively charged phospholipids.

In some embodiments, the biomimetic membranes of the invention compriseone or more reactive phospholipids. Examples of reactive phospholipidsinclude, but are not limited to, phosphatidyl ethanolamine derivativescoupled to a polyethyleneglycol, a biotinyl, a glutaryl, a caproyl, amaleimide, a sulfhydral, a pyridinal disulfide or a succinyl amine. Insome embodiments, the biomimetic membranes of the invention comprise atleast 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 70%, 80%, 90% or 100% of reactive phospholipids. In someembodiments, the biomimetic membranes of the invention comprise at least2% of reactive phospholipids. In some embodiments, the biomimeticmembranes of the invention comprise at least 5% of reactivephospholipids. In some embodiments, the biomimetic membranes of theinvention comprise at least 10% of reactive phospholipids. In someembodiments, the biomimetic membranes of the invention comprise at least25% of reactive phospholipids. In some embodiments, the biomimeticmembranes of the invention comprise at least 30% of reactivephospholipids.

In some embodiments, the biomimetic membranes of the invention compriseone or more lipids and phospholipids such as soy lecithin, partiallyrefined lecithin, hydrogenated phospholipids, lysophosphate,phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,phosphatidylinositol, cardiolipin, sphingolipids, gangliosides,cerebrosides, ceramides, other esters analogue of phopshpatidylcholine(PAF, lysoPAF). In some embodiments, the biomimetic membranes of theinvention comprise one or more synthetic phospholipids such asL-a-lecithin (dilauroylphosphatidylcholine,dipalmitoylphosphatidylcholine, dilinoloylphosphatidylcholine,distearoylphosphatidylcholine, diarachidoylphosphatidylcholine);phosphatidylethanolamine derivatives, such as1,2-diacyl-sn-glycero-3-phosphoethanolamine,1-acyl-2-acyl-sn-glycero-3-phosphoethanolamine, dinitrophenyl- anddinitrophenylamino caproylphosphatidylethanolamine,1,2-diacyl-sn-glycero-3-phosphoethanolamine-N-poly ethylene glycol(PEG-PE), N-biotinyl-PE, N-caproylamine PE, N-dodecylamine-PE, N-MPB-PE,N-PDD-PE, N-succinyl-PE, N-glutaryl-PE; di-acetylenic lipids;phosphatidic acids (1,2-diacyl-sn-glycero-3-phosphate salt,1-acyl-2-acyl-sn-glycero-3-phosphate sodium salt; phosphatidylserinesuch as 1,2-diacyl-snglycero-3-[phospho-L-serine] sodium salt,1-acyl-2-acyl-sn-glycero-3-[phospho-L-serine] sodium salt,lysophosphatidic acid; cationic lipids such as1,2-diacyl-3-trimethylammoniumpropane (TAP),1,2-diacyl-3-dimethylammoniumpropane (DAP), N-[1-(2,3-dioleoyloxy)propyl-N,N′,N″-trimethylammonium chloride (DOTMA).

In some embodiments, the biomimetic membranes of the invention compriseone or more lipids suitable for click chemistry, such as thosecontaining azide and alkyne groups. In some embodiments, the biomimeticmembranes of the invention comprise one or more phospholipids withmultivarious headgroups such as phosphatidylethanol,phosphatidylpropanol and phosphatidylbutanol,phosphatidylethanolamine-N-monomethyl,1,2-disteraoyl(dibromo)-sn-glycero-3-phosphocoline. In some embodiments,the biomimetic membranes of the invention comprise one or morephospholipids with partially or fully fluorinated cholesterol orcholesterol derivatives can be used in place of an uncharged lipid, asgenerally known to a person skilled in the art.

The surface of a biomimetic membrane may also be modified with apolymer, such as, for example, with polyethylene glycol (PEG), usingprocedures readily apparent to those skilled in the art. Lipids maycontain functional surface groups for attachment to a metal, whichprovides for the chelation of radioactive isotopes or other materialsthat serve as the therapeutic entity. Any species of lipid may be used,with the sole proviso that the lipid or combination of lipids andassociated materials incorporated within the lipid matrix should form amonolayer phase under physiologically relevant conditions. As oneskilled in the art will recognize, the composition of the biomimeticmembrane may be altered to modulate the biodistribution and clearanceproperties of the resulting biomimetic membranes.

Other useful lipids or combinations thereof apparent to those skilled inthe art which are in keeping with the spirit of the present inventionare also encompassed by the present invention. For example,carbohydrates bearing lipids may be employed for in vivo targeting asdescribed in U.S. Pat. No. 4,310,505.

In some embodiments, the biomimetic membranes of the invention compriseone or more polymerizable lipid. Polymerizable lipids that can be usedin the present invention include those described in U.S. Pat. Nos.5,512,294 and 6,132,764, and US publication No. 2010/0111840,incorporated by reference herein in their entirety.

In some embodiments, the hydrophobic tail groups of polymerizable lipidsare derivatized with polymerizable groups, such as diacetylene groups,which irreversibly cross-link, or polymerize, when exposed toultraviolet light or other radical, anionic or cationic, initiatingspecies, while maintaining the distribution of functional groups at thesurface of the biomimetic membrane. The resulting polymerized biomimeticmembrane is stabilized against fusion with cell membranes or otherbiomimetic membranes and stabilized towards enzymatic degradation. Thesize of the polymerized biomimetic membranes can be controlled by themethod described herein, but also by other methods known to thoseskilled in the art, for example, by extrusion.

Polymerized biomimetic membranes may be comprised of polymerizablelipids, but may also comprise saturated and non-alkyne, unsaturatedlipids. The polymerized biomimetic membranes can be a mixture of lipidswhich provide different functional groups on the hydrophilic exposedsurface. For example, some hydrophilic head groups can have functionalsurface groups, for example, biotin, amines, cyano, carboxylic acids,isothiocyanates, thiols, disulfides, α-halocarbonyl compounds,α,β-unsaturated carbonyl compounds and alkyl hydrazines. These groupscan be used for attachment of targeting agents, such as antibodies,ligands, proteins, peptides, carbohydrates, vitamins, nucleic acids orcombinations thereof for specific targeting and attachment to desiredcell surface molecules, and for attachment to other surfaces. Otherhydrophilic head groups can have a functional surface group ofdiethylenetriamine pentaacetic acid, ethylenedinitrile tetraacetic acid,tetraazocyclododecane-1,4,7,10-tetraacetic acid (DOTA), porphoryinchelate and cyclohexane-1,2,-diamino-N,N′-diacetate, as well asderivatives of these compounds, for attachment to a metal, whichprovides for the chelation of radioactive isotopes or other materials.Examples of lipids with chelating head groups are provided in U.S. Pat.No. 5,512,294, incorporated by reference herein in its entirety.

The component lipids of the biomimetic membranes can be purified andcharacterized individually using standard, known techniques and thencombined in controlled fashion to produce the final particle. Thebiomimetic membranes can be constructed to mimic native cell membranesor present functionality, such as ethylene glycol derivatives.Additionally, the biomimetic membranes can have a well-defined monolayeror bilayer structure that can be characterized by known physicaltechniques such as transmission electron microscopy and atomic forcemicroscopy.

b. Polymers

In one aspect, the biomimetic membranes of the invention comprise one ormore polymers. In some embodiments, the biomimetic membranes compriseblock copolymer membranes simulating a natural environment. In someembodiments, the polypeptide(s) described herein (e.g. aquaporin and/orhydrophobin) are incorporated into the block copolymer membranessimulating a natural environment.

Various types of amphiphilic copolymers can be used. In one embodiment,the copolymer is an ABA copolymer, where A is hydrophilic and B ishydrophobic where A is the same or different hydrophilic segments and Bis a hydrophobic B segment. Thus, the term “ABA copolymer” includes anABC copolymer, where the hydrophilic segments A and C are different.

One class of suitable polymeric materials is described in U.S. Pat. No.5,807,944 to Hirt, et al. In one embodiment, the amphiphilic polymer isbiocompatible. Many suitable amphiphilic copolymers and hydrophobic andhydrophilic copolymers are described in WO 97/49387.

The block copolymer includes at least one segment B that includes ahydrophobic polymer. Any of a number of hydrophobic polymers can beused, such as, but not limited to, polysiloxane such aspolydimethylsiloxane and polydiphenylsiloxane, perfluoropolyether,polystyrene, polyoxypropylene, polyvinylacetate, polyoxybutylene,polyisoprene, polybutadiene, polyvinylchloride, polyalkylacrylate (PAA),polyalkylmethacrylate, polyacrylonitrile, polypropylene, PTHF,polymethacrylates, polyacrylates, polysulfones, polyvinylethers, andpoly(propylene oxide), and copolymers thereof.

In some embodiments, the hydrophobic segment contains a predominantamount of hydrophobic monomers. A hydrophobic monomer is a monomer thattypically gives a homopolymer that is insoluble in water and can absorbless than 10% by weight of water.

Suitable hydrophobic monomers are C1-C18 alkyl and C3-C18 cycloalkylacrylates and methacrylates, C3-C18 alkylacrylamides and-methacrylamides, acrylonitrile, methacrylonitrile, vinyl C1-C18alkanoates, C2-C18 alkenes, C2-C18 haloalkenes, styrene, (loweralkyl)styrene, C4-C12 alkyl vinyl ethers, C2-C10 perfluoro-alkylacrylates and methacrylates and correspondingly partially fluorinatedacrylates and methacrylates, C3 through C12perfluoroalkylethylthiocarbonylaminoethyl acrylates and methacrylates,acryloxy- and methacryloxyalkylsiloxanes, N-vinylcarbazole, C1 throughC12 alkyl esters of maleic acid, fumaric acid, itaconic acid, mesaconicacid, vinyl acetate, vinyl propionate, vinyl butyrate, vinyl valerate,chloroprene, vinyl chloride, vinylidene chloride, vinyltoluene, vinylethyl ether, perfluorohexyl ethylthiocarbonylaminoethyl methacrylate,isobornyl methacrylate, trifluoroethyl methacrylate,hexa-fluoroisopropyl methacrylate, hexafluorobutyl methacrylate,tristrimethylsilyloxysilylpropyl methacrylate (TRIS), and3-methacryloxypropylpentamethyldisiloxane.

In some embodiments, the hydrophobic polymer is one which displays arelatively high oxygen diffusion rate there through, such as, but notlimited to, polysiloxanes, perfluoroalkyl ethers, specific unsaturatedpolymers, and polysulfones. In one embodiment, the hydrophobic polymeris a polysiloxane block having terminal alkylene groups.

In some embodiments, the hydrophobic polymer includes aperfluoroalkyl-polyether block. In other embodiments, the hydrophobicpolymer includes an unsaturated polymer, such as a polymer of aconjugated aliphatic or alicyclic diene, which may be substituted byhalogen or lower alkyl, a polymer of an alkyne or dialkyne, which may besubstituted by lower alkyl or trimethylsilyl, a copolymer of aconjugated diene and a hydrophilic or hydrophobic vinylic monomer, andalso partially hydrated derivatives of these compounds.

Specific examples of polymers of conjugated dienes are cis-, trans-,iso- or syndiotactic poly-1,2-butadiene, poly-1,4-butadiene orpolyisoprene, poly-pentenamer, polychloroprene and polypiperylen. Otherexamples of copolymers are butadiene- or isoprene-copolymers withhydrophilic or hydrophobic vinylic monomers, such as acrylonitrile,styrene, acrylic acid or hydroxyethylmethacrylate. An example of apolyalkyne is poly-1-trimethylsilyl-propyne. In some embodiments,examples of polymers included unsaturated polymers are syndiotacticpoly-1,2-butadiene, poly-1,4-butadiene and polyisoprene. An especiallypreferred unsaturated polymer is poly-1-trimethylsilyl-propyne. Anotherespecially preferred unsaturated polymer is poly-1,4-butadiene.

The hydrophobic polymer may include a single type of polymer or morethan one type of polymer, such as two or more of those discussed above.The mean molecular weight of one segment B is in the range from about500 to about 50,000, preferably in the range from about 800 to about15,000, more preferably in the range of about 1,000 to 12,000,particularly preferably in the range from about 5,000 to about 12,000.

In some embodiments, in addition to the hydrophobic segment B, theamphiphilic segmented copolymer includes at least one segment A whichincludes at least one hydrophilic polymer, such as, but not limited to,polyoxazoline, polyethylene glycol, polyethylene oxide, polyvinylalcohol, polyvinylpyrrolidone, polyacrylamide, poly(meth)acrylic acid,polyethylene oxide-co-polypropyleneoxide block copolymers,poly(vinylether), poly(N,N-dimethylacrylamide), polyacrylic acid,polyacyl alkylene imine, polyhydroxyalkylacrylates such as hydroxyethylmethacrylate (HEMA), hydroxyethyl acrylate, and hydroxypropyl acrylate,polyols, and copolymeric mixtures of two or more of the above mentionedpolymers, natural polymers such as polysaccharides and polypeptides, andcopolymers thereof, and polyionic molecules such as polyallylammonium,polyethyleneimine, polyvinylbenzyltrimethylammonium, polyaniline,sulfonated polyaniline, polypyrrole, and polypyridinium,polythiophene-acetic acids, polystyrenesulfonic acids, zwitterionicmolecules, and salts and copolymers thereof.

In some embodiments, the hydrophilic segment preferably contains apredominant amount of hydrophilic monomers. A hydrophilic comonomer is amonomer that typically gives a homopolymer that is soluble in water orcan absorb at least 10% by weight of water.

Suitable hydrophilic monomers are hydroxyl-substituted lower alkylacrylates and methacrylates, acrylamide, methacrylamide, (lower alkyl)acrylamides and methacrylamides, N,N-dialkyl-acrylamides, ethoxylatedacrylates and methacrylates, polyethyleneglycol-mono methacrylates andpolyethyleneglycolmonomethylether methacrylates, hydroxyl-substituted(lower alkyl)acrylamides and methacrylamides, hydroxyl-substituted loweralkyl vinyl ethers, sodium vinylsulfonate, sodium styrenesulfonate,2-acrylamido-2-methylpropanesulfonic acid, N-vinylpyrrole,N-vinyl-2-pyrrolidone, 2-vinyloxazoline,2-vinyl-4,4′-dialkyloxazolin-5-one, 2- and 4-vinylpyridine, vinylicallyunsaturated carboxylic acids having a total of 3 to 5 carbon atoms,amino(lower alkyl)-(where the term amino also includes quaternaryammonium), mono(lower alkylamino)(lower alkyl) and di(loweralkylamino)(lower alkyl) acrylates and methacrylates, allyl alcohol,3-trimethylammonium 2-hydroxypropylmethacrylate chloride (Blemer, QA,for example from Nippon Oil), dimethylaminoethyl methacrylate (DMAEMA),dimethylaminoethylmethacrylamide, glycerol methacrylate, andN-(1,1-dimethyl-3-oxobutyl)acrylamide.

In some embodiments, the segment A includes a polymer displaying arelatively high water or ion diffusion rate there through. Specificexamples of hydrophilic monomers from which such polymers can be madeare cyclic imino ethers, vinyl ethers, cyclic ethers including epoxides,cyclic unsaturated ethers, N-substituted aziridines, beta-lactones andbeta-lactames. Further suitable monomers include ketene acetals, vinylacetals and phosphoranes. Suitable cyclic imino ethers include2-oxazoline. If a 2-oxazoline having an alkenyl group in 2 position isused as hydrophilic monomer, a polymerizable unsaturated group isprovided within segment A (in a side chain) of the amphiphilic segmentedcopolymer to serve as the polymerizable unsaturated group necessary forthe final polymerization to obtain a polymeric product or as anadditional polymerizable unsaturated group which offers the possibilityof direct crosslinking in the preparation of the polymer. In someembodiments, the cyclic imino ether is 2-methyloxazoline. The mostpreferred vinyl ethers are methyl vinyl ether, ethyl vinyl ether andmethoxy ethyl vinyl ether.

In some embodiments, the mean molecular weight of one segment A is inthe range from about 500 to about 50,000, from about 800 to about15,000, from about 1,000 to 12,000, particularly from about 5,000 toabout 12,000.

The mixtures of block copolymers can be mixtures of two or more of thefollowing classes, where the separate components can be of the sameclass but with a different distribution of polymer blocks:

Polymer source triblock copolymers E/EP/E, of poly(ethylene)(E) andpoly(ethylenepropylene) (EP) triblock copolyampholytes. Among (N,Ndimethylamino)isoprene, such polymers are Ai14S63A23, Ai31S23A46,Ai42S23A35, styrene, and methacrylic acid Ai56S23A21, Ai57S11A32.

Styrene-ethylene/butylene-styrene (KRATON) G 1650, a 29% styrene, 8000solution triblock copolymer viscosity (25 wt-% polymer), 100% triblockstyrene-ethylene/butylene-styrene (S-EB-S) block copolymer; (KRATON) G1652, a 29% styrene, 1350 solution viscosity (25 wt-% polymer), 100%triblock S-EB-S block copolymer; (KRATON) G 1657, a 4200 solutionviscosity (25 wt-% polymer), 35% diblock S-EB-S block copolymer; allavailable from the Shell Chemical Company. Such block copolymers includethe styrene-ethylene/propylene (S-EP) types and are commerciallyavailable under the tradenames (KRATON) G 1726, a 28% styrene, 200solution viscosity (25 wt-% polymer), 70% diblock S-EB-S blockcopolymer; (KRATON) G-1701X a 37% styrene, >50,000 solution viscosity,100% diblock S-EP block copolymer; and (KRATON) G-1702X, a 28%styrene, >50,000 solution viscosity, 100% diblock SEP block copolmyer.

Siloxane triblock copolymer PDMS-b-PCPMS-b-PDMSs(PDMS=polydimethylsiloxane, PCPMS=poly(3-cyanopropylmethylsiloxane) canbe prepared through kinetically controlled polymerization ofhexamethylcyclotrisiloxane initiated by lithium silanolate endcappedPCPMS macroinitiators. The macroinitiators can be prepared byequilibrating mixtures of 3-cyanopropylmethylcyclosiloxanes (DXCN) anddilithium diphenylsilanediolate (DLDPS). DxCNs can be synthesized byhydrolysis of 3-cyanopropylmethyldichlorosilane, followed by cyclizationand equilibration of the resultant hydrolysates. DLDPS can be preparedby deprotonation of diphenylsilanediol with diphenylmethyllithium.Mixtures of DXCN and DLDPS can be equilibrated at 100° C. within 5-10hours. By controlling the DxCN-to-DLDPS ratio, macroinitiators ofdifferent molecular weights are obtained. The major cyclics in themacroinitiator equilibrate are tetramer (8.6+−0.7 wt %), pentamer(6.3+−0.8 wt %) and hexamer (2.1+−0.5 wt %).

2.5 k-2.5 k-2.5 k, 4 k-4-k-4-k, and 8 k-8 k-8 k triblock copolymers havebeen characterized. These triblock copolymers are transparent,microphase separated and highly viscous liquids. PEO-PDMS-PEO triblockformed from Polyethylene oxide (PEO) and poly-copolymer dimethylsiloxane (PDMS). Functionalized poly (2 methyloxazoline)-block-: TheseA-B-A polymers include poly(dimethylsiloxane)-block-versions in whichthe A components have MW of poly(2-methyloxazoline) triblockapproximately 2 kDa, and the B component of copolymer approximately 5kDa, and (b) the A components have MW of approximately 1 kDa, and the Bcomponent of approximately 2 kDa. Poly(d/1-lactide) (“PLA”)-PEG-PLAtriblock copolymer. Poly(styrene-b-butadiene-b-styrene) triblockcopolymer.

Poly(ethylene (such polymers included Pluronic F127, Pluronic P105, oroxide)/poly(propylene oxide) Pluronic L44 from BASF (PerformanceChemicals). Triblock copolymers PDMS-PCPMS-PDMS. A series of epoxy andvinyl end-capped polysiloxane (polydimethylsiloxane-triblock copolymerswith systematically varied molecular polycyanopropylmethylsiloxane)weights can be synthesized via anionic polymerization triblock copolymerusing LiOH as an initiator. Polydiene-polystyrene-polydiene available asProtolyte A700 from DAIS-Analytic, Odessa, Fla. Azo-functionalstyrene-butadiene-HEMA triblock copolymer. Amphiphilic triblockcopolymer carrying polymerizable end groups. Syndiotacticpolymethylmethacrylate (sPMMA)-polybutadiene (PBD)-sPMMA triblockcopolymer. Tertiary amine methacrylate triblock BiodegradablePLGA-b-PEO-b-PLGA triblock copolymer,Polyactide-b-polyisoprene-b-polyactide triblock copolymer,Poly(isoprene-block-styrene-block-dimethylsiloxane) triblock copolymer,Poly(ethylene oxide)-block-polystyrene-block-poly(ethylene oxide)triblock copolymer, Poly(ethylene oxide)-poly(THF)-poly(ethylene oxide)triblock copolymer. Ethylene oxide triblock Poly E-caprolactone(Birmingham Polymers, Birmingham), AL Poly(DL-lactide-co-glycolide)(Birmingham Polymers), Poly(DL-lactide) (Birmingham Polymers),Poly(L-lactide) (Birmingham Polymers), Poly(glycolide) (BirminghamPolymers), Poly(DL-lactide-co-caprolactone) (Birmingham Polymers),Styrene-Isoprene-styrene triblock (Japan Synthetic Rubber Co., Tokyo,Japan) MW=140 kg/mol, copolymer Block ratio of PS/PI=15/85.PMMA-b-PIB-b-PMMA Poly(methyl methacrylate) (PMMA) and polyisobutylene(PIB). PLGA-PEO-PLGA triblock Polymers of poly(DL-lacticacid-co-glycolic acid) copolymer (PLGA) and PEO. Sulfonatedstyrene/ethylene-butylene/styrene (S-SEBS) triblock copolymer protonconducting membrane. Poly(1-lactide)-block-poly(ethyleneoxide)-block-poly(1-lactide) triblock copolymer Poly-ester-ester-estertriblock copolymer PLA/PEO/PLA triblock copolymer. The synthesis of thetriblock copolymers can be prepared by ring-opening polymerization ofDL-lactide or e-caprolactone in the presence of poly(ethylene glycol),using no-toxic Zn metal or calcium hydride as co-initiator instead ofthe stannous octoate. The composition of the co-polymers can be variedby adjusting the polyester/polyether ratio.

The above polymers can be used in mixtures of two or more of polymers inthe same or different class. For example, in two polymer mixturesmeasured in weight percent of the first polymer, such mixtures cancomprise 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45% or45-50%. Or, for example where three polymers are used: the first cancomprise 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45% or45-50% of the whole of the polymer components, and the second can10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45% or 45-50% of theremainder.

Preparation of the Amphiphilic Copolymer

In some embodiments, the segments A and B are linked together through abond that may be hydrolyzable or non-hydrolyzable. A non-hydrolyzablebond is a covalent bond that is not cleaved by an ordinary aqueous orsolvent hydrolysis reaction, e.g. under acidic or basic conditions.Specific bonds that are hydrolyzable are well known to those skilled inthe art.

A non-hydrolyzable bond between segments A and B in the amphiphilicsegmented copolymer can be formed by polymerizing a suitable hydrophilicmonomer (from segment A) in the presence of a suitably functionalizedhydrophobic monomer (from segment B) such that a block of units of thehydrophilic monomer grows from the site of functionalization of thehydrophilic monomer or, alternatively by polymerizing a suitablehydrophobic monomer in the presence of a suitably functionalizedhydrophilic monomer such that a block of units of the hydrophobicmonomer grows from the site of functionalization of the hydrophilicmonomer.

The functionalized segment is also called a macroinitiator. Suitablemacroinitiators include thermally or photochemically activatablecationic or anionic groups, or a thermally or photochemicallyactivatable radical initiator group. Anionic polymerization,polycondensation, and polyaddition can also be used. Specific examplesof preferred photochemically activatable cationic initiator groups aretriflate (—O—SO₂—CF₃), —I (iodide), —O-mesyl, —O-tosyl, and —Cl+AgSbF₆.In some embodiments, the preferred initiator group is the triflategroup. The initiator group is linked to the starting segment in a waythat provides a covalent non-hydrolyzable bond between the terminalgroup of the starting segment and the first monomer forming the growingsegment that is attached to the starting segment during the graftcopolymerization for preparing the amphiphilic segmented copolymer.Grafting means that polymer chains are grown from a monomer either interminal or in pendant position onto another preformed polymer.

The initiator group may be introduced into a preformed polymer in asuitable way, for example through linkage of cationic or thermalinitiator groups to functional groups present on the starting monomer.Only the latter method is suitable for providing pendent initiatorgroups. Preferred triflate groups can be introduced by reaction ofterminal or pendent functional hydroxyl groups with activated triflicacid derivatives such as (CF₃SO)₂O.

A degradable bond between the A segment and the B segment can be used sothat the biomimetic membrane (e.g. vesicle) can be degraded. Degradablebonds within the A or B segment can also be used. Biodegradable regionscan be constructed from polymers or monomers using linkages susceptibleto biodegradation. Degradability can be imparted by inclusion of asingle degradable linkage or a degradable region made of more than onedegradable linkage. The terms degradable linkage and degradable regionare used interchangeably hereinafter.

The degradable region is preferably degradable under in vivo conditions.For example, a degradable region may be a hydrolyzable region, such asmade from a polymer or oligomer of glycolide, lactide,epsilon-caprolactone, other hydroxy acids, or other biologicallydegradable polymer that yields materials that are non-toxic or presentas normal metabolites in the body. Regions that are biodegradable byenzymatic degradation can also be used. Preferred poly(alpha-hydroxyacids) are poly(glycolic acid), poly(DL-lactic acid), and poly(L-lacticacid). Other useful materials include poly(amino acids),poly(anhydrides), poly(orthoesters), poly(phosphazines) andpoly(phosphoesters). Polylactones such as poly(epsilon-caprolactone),poly(delta-valerolactone), and poly(gamma-butyrolactone), for example,are also useful. The biodegradable region may have a degree ofpolymerization ranging from one up to values that would yield a productthat is not substantially water soluble. Thus, monomeric, dimeric,trimeric, oligomeric, and polymeric regions may be used.

Accordingly, the amphiphilic segmented copolymers may consist in oneembodiment of one segment A and one segment B (A-B-type, diblock), or ofone segment A and two segments B attached to its termini (B-A-B-type,triblock), or may have a comb-type structure wherein several segments Bare pendent from one segment A, which may further carry one or twoterminal segments B). In another embodiment, the amphiphilic segmentedcopolymers may consist of one segment B and two segments A attached toits termini (A-B-A-type, triblock). In another embodiment, theamphiphilic segmented copolymers may have a comb-type structure whereinseveral segments A are pendent from one segment B, which may furthercarry one or two terminal segments A. In some embodiments, the copolymeris an ABA or ABC triblock copolymer.

It is also possible to change the monomer during graft copolymerizationsuch that, for example, first hydrophilic segments A are grown on apreformed hydrophobic segment B and then hydrophobic segments B′ areattached to the termini of the earlier prepared segments A. Also adifferent hydrophilic monomer may be used to produce a differenthydrophilic segment A′ at the termini of the hydrophilic segments A.Again, other embodiments of the amphiphilic segmented copolymers may beproduced starting from a functionalized hydrophilic segment A.

In some embodiments, the polymer that makes up the starting segment (Aor B) usually has a number average molecular weight Mn in the range fromabout 500 to about 50,000, from about 800 to about 15,000, about 1,000to 12,000, from about 5,000 to about 12,000. The length of the one ormore segments A, B, A′, or B′ which are to be graft copolymerized on thestarting segment can be easily controlled by controlling the amount ofmonomer (hydrophilic or hydrophobic) which is added for the graftcopolymerization. In this way the size of the segments and their ratiocan easily be controlled.

The amphiphilic segmented copolymers can be prepared in the presence orabsence of a solvent. It is advantageous to use a substantially inertsolvent, i.e. one that does not participate in the reaction. Suitableexamples are halogenated hydrocarbons, such as chloroform or methylenechloride, bipolar aprotic solvents, such as acetonitrile, acetone,dimethylformamide (DMF) or dimethyl sulfoxide (DMSO), hydrocarbons, suchas toluene or xylene, and pyridine or N-methylmorpholine, and mixturesthereof.

In the preparation of the amphiphilic segmented copolymers of theinvention, the reaction temperature can be, for example, from −60° C. to150° C., preferably from 0° C. to 80° C. The reaction times are in therange from about 15 minutes to 7 days, preferably in the region of about2 to 48 hours. If necessary, the reaction is carried out under argon ornitrogen as protective gas. A suitable catalyst, for example dibutyltindilaurate (DBTDL), is added in the urethane-forming terminalfunctionalizing reaction.

In some embodiments, the biomimetic membrane can be crosslinked toprovide additional stability. Crosslinking can be achieved using manystandard techniques, including photopolymerization, for example, ofacrylate groups in the presence of a photoinitiator, or through the useof an alkylating agent. Crosslinking can also be achieved using sidegroups and end groups which can be polymerized by free radicalpolymerization, side groups which can be polymerized by cationicpolymerization, and side groups which can be polymerized by ring-openingpolymerization.

In addition to the hydrophilic and hydrophobic segments, the membranesmay also include additional hydrophobic and/or hydrophilic components,as well as crosslinkers such as monomers or macromers with reactivegroups, surfactants, and crosslinking initiators, especiallyphotoinitiators. Targeting or biological signal molecules can beattached to the outside surface of the biomimetic membranes (e.g.vesicles).

Polymerization Groups

The segmented copolymers may already contain polymerizable groups in thehydrophobic and/or hydrophilic segments, e.g. if a hydrophobic segment Bcomprises a dienepolymer like polybutadiene or polyisoprene, or if themonomer used for making a hydrophilic segment comprises an unsaturatedside chain, for example 2-allyl-oxazoline. Whether or not present, it ispossible to introduce polymerizable groups by suitable reactions, e.g.at the end of or pendent from the growing segments. For this purpose,the graft polymerization of the growing segment may be terminated aftera suitable chain length is reached and the initiator group present atthe chain end capped, for example, either by using specific reagentssuch as hydroxy styrene, allyl alcohol, HEMA, propargyl alcohol, allylamines and propargyl amine, or by using KOH/EtOH or primary aminesleaving —OH or —NH— groups or unsaturated groups at the end of thegrowing segment. Hydroxyl groups may also be introduced into thecopolymers by employing suitable comonomers in the graftcopolymerization, e.g. 2-hydroxy-alkyloxazolines. The hydroxyl or —NH—groups may then be reacted, e.g. with an isocyanate carrying apolymerizable unsaturated group. Preferred examples of such bifunctionalcompounds are 2-isocyanatoethyl methacrylate (IEM), which is especiallypreferred, and vinyl isocyanate, allyl isocyanate, acryloyl isocyanate,styrene isocyanate, vinyl benzyl isocyanate, propargyl isocyanate, and(meth)acrylic anhydride. Other polymerizable groups can be introduced bymethods known to those skilled in the art.

Any type of polymerization/crosslinking can be used. Examples includephotopolymerization, redox polymerization, anionic polymerization,condensation reactions, addition reactions, and chain polymerizationreactions.

Additional Monomers

In some embodiments, the proportion by weight of the amphiphilicsegmented copolymer in the biomimetic membrane is in the range from 100to 50%, in particular in the range from 100 to 80%, preferably in therange from 100 to 90%, based on the total polymeric product. Thepolymeric biomimetic membrane may be obtained by direct thermal orphotochemical polymerization or crosslinking reaction of the amphiphilicsegmented copolymer without the addition of comonomers in the presenceof a suitable initiator. However, in some cases, it may be preferable toinclude a comonomer. Types of comonomers that may be desired includehydrophobic or hydrophilic comonomers, or cationic or anioniccomonomers. It may also be desirable to include a comonomer thatcontains a specific functional group, such as a crosslinkable group, ora group that has a particular affinity for a molecule to be incorporatedinto or onto the membrane, as discussed below. Suitable hydrophobic andhydrophilic comonomers include those discussed above.

The comonomers can be included within the amphiphilic polymer network,or crosslinked as an interpenetrating or semi-interpenetrating networkwith the amphiphilic polymer. Crosslinking may be achieved with theaddition of a comonomer and/or a crosslinking agent, for example, apolyunsaturated comonomer.

Crosslinking Agents

A polymer network can, if desired, be reinforced by addition of acrosslinking agent, for example, a polyunsaturated comonomer. Acrosslinked biomimetic membrane including the product of thepolymerization and crosslinking reaction of an amphiphilic segmentedcopolymer, can also be formed, if desired, with at least one vinyliccomonomer and with at least one crosslinking agent. Crosslinking can beachieved by a number of different means, such as but not limited to,free radical crosslinking, redox crosslinking, and salt crosslinking.

Examples of suitable crosslinking agents include allyl methacrylate,lower alkylene glycol dimethacrylate, poly(lower alkylene)glycoldimethacrylate, lower alkylene dimethacrylate, divinyl ether, divinylsulfone, di- and trivinylbenzene, trimethylolpropane trimethacrylate,pentaerythritol tetramethacrylate, bisphenol A dimethacrylate,methylenebis(meth)acrylamide, triallyl phthalate and diallylphthalate,.alpha.-.omega.-bis(methacryloxyalkyl)-oligosiloxanes such asbis(methacryloxypropyl)tetramethyldisiloxane, and perfluoroalkyl- orperfluoroalkylether-bismethacrylates.

The amount of crosslinking agent used is expressed in a proportion byweight based on the total polymer and is in the range from 20 to 0.05%,in particular in the range from 10 to 0.1%, preferably in the range from5 to 0.1%.

Crosslinking Initiators

The biomimetic membranes are crosslinked in a manner known in the artfrom the corresponding monomers (the term monomer here also including anamphiphilic segmented copolymer) by a polymerization reaction customaryto the person skilled in the art.

In the case of monomers that can be crosslinked with free radicalcrosslinking, a mixture of monomers is typically warmed with addition ofa free-radical former. Examples of such free-radical formers areazoisobutyronitrile (AIBN), potassium peroxodisulfate, dibenzoylperoxide, hydrogen peroxide, and sodium percarbonate. If, for example,the compounds are warmed, free radicals form with homolysis, and canthen initiate polymerization.

A polymerization reaction may be carried out using a photoinitiator thatcan initiate free-radical polymerization and/or crosslinking. Examplesof suitable photoinitiators include benzoin methyl ether,1-hydroxycyclohexylphenyl ketone, and Darocure and Irgacure products,preferably Darocure 1173®, and Irgacure 2959®. Also suitable arereactive photoinitiators, which can be incorporated, for example, into amacromer, or can be used as a specific comonomer. Examples are describedin European Patent No. EP 0 632 329. The photopolymerization can then beinitiated by actinic radiation, for example light, in particular UVlight having a suitable wavelength. The spectral requirements can, ifnecessary, be controlled appropriately by addition of suitablephotosensitizers.

The polymerizable regions may be polymerizable by photoinitiation byfree radical generation, most preferably in the visible or longwavelength ultraviolet radiation. Polymerizable regions are acrylates,diacrylates, oligoacrylates, dimethacrylates, oligomethoacrylates, orother biologically acceptable photopolymerizable groups. A preferredtertiary amine is triethanol amine.

Useful photoinitiators are those that can be used to initiate by freeradical generation polymerization of the macromers without cytotoxicityand within a short time frame, minutes at most and most preferablyseconds. Preferred dyes as initiators of choice for LWUV initiation areethyl eosin, 2,2-dimethoxy-2-phenyl acetophenone, other acetophenonederivatives, and camphorquinone. In all cases, crosslinking andpolymerization are initiated among copolymers by a light-activatedfree-radical polymerization initiator such as2,2-dimethoxy-2-phenylacetophenone or a combination of ethyl eosin(10⁻⁴-10² milliM) and triethanol amine (0.001 to 0.1 M), for example.

The choice of the photoinitiator is largely dependent on thephotopolymerizable regions. For example, when the macromer includes atleast one carbon-carbon double bond, light absorption by the dye causesthe dye to assume a triplet state, the triplet state subsequentlyreacting with the amine to form a free radical that initiatespolymerization. Preferred dyes for use with these materials includeeosin dye and initiators such as 2,2-dimethyl-2-phenylacetophenone,2-methoxy-2-phenylacetophenone, and camphorquinone. Using suchinitiators, copolymers may be polymerized in situ by long wavelengthultraviolet light or by laser light of about 514 nm, for example. Lasersmay be used to polymerize any nanospheres from a photopolymerizablesolution, due to the precise control that can be achieved with thelasers. It is thus possible to make nanospheres as described hereinwithout inclusion of the amphiphilic polymers.

Initiation of polymerization is accomplished by irradiation with lightat a wavelength of between about 200-700 nm, most preferably in the longwavelength ultraviolet range or visible range, 320 nm or higher, mostpreferably about 514 nm or 365 nm. The light-sensitive free-radicalpolymerization initiator may be a single compound (e.g.2,2-dimethoxy-2-phenyl acetophenone) or a combination of a dye and acocatalyst (e.g., ethyl eosis and triethanol amine).

Solvents

Polymerization/crosslinking can be carried out in the presence orabsence of a solvent. Suitable solvents are all solvents which dissolvethe monomers used, for example, water, alcohols such as lower alkanolslike ethanol or methanol, carboxamides such as dimethylformamide,dipolar aprotic solvents such as dimethyl sulfoxide or methyl ethylketone, ketones such as acetone or cyclohexanone, hydrocarbons such astoluene, ethers such as THF, dimethoxyethane or dioxane, halogenatedhydrocarbons such as trichloroethane, and mixtures of suitable solventssuch as mixtures of water and an alcohol, for example, a water/ethanolor water/methanol mixture.

c. Preparation of Biomimetic membranes

In some embodiments, the polypeptide(s) (e.g. aquaporins and/orhydrophobin) are been reconstituted in biomimetic membranes that arevesicles (e.g., polymer or lipid (e.g. liposomes, micelles,nanoparticles, microbubbles, etc. . . . )) prepared by methods known inthe art. In some embodiments, the vesicles are supported into asubstrate. In some embodiments, the lipid vesicles are transformed intoa supported lipid bilayer membranes using a method known in the art suchas the Langmuir-Blodgett method. In some embodiments, the vesicles ofthe invention are coated with one or more hydrophobins as describedherein.

In some embodiments, the biomimetic membranes can be formed from lipidor polymer solutions. In some embodiments, the lipid or polymersolutions can be prepared using suitable protocols known in the art.

In some embodiments, the polypeptide(s) (e.g. aquaporins and/orhydrophobin) are been reconstituted in biomimetic membranes that arelipid vesicles, and transformed into a supported lipid bilayer membranesusing a method such as the Langmuir-Blodgett method. Intrinsicpermeability of the membrane material must be secured. A material withlow permeability can be used, however, it must at the same time berobust and able to incorporate the polypeptide(s) to constitute overalla stable and dense 2D filtering array. Various procedures are commonlyused for preparing supported bilayers. A simple technique is theLangmuir-Blodgett method. A solution of lipid in a suitable organicsolvent is spread on an aqueous sub phase in a Langmuir trough and theorganic solvent is evaporated. A pair of movable barriers is used tocompress the lipid film laterally to a desired surface pressure. Thenthe substrate is passed vertically through the film thereby transferringa one molecule thick lipid layer (monolayer) onto the substrate. Asecond monolayer can be transferred by passing the substrate through thefilm once more. A total of three monolayers have been transferred by thevertical (Langmuir-Blodgett) deposition method, however, a fourth layermay be transferred by using horizontal, the so called Langmuir-Schaeffer(LS), deposition for the last layer. The methods can be used with avariety of lipids, including those described herein. Native biologicalmembranes often are asymmetric. Both LB and LS offer the possibility ofpreparing asymmetric bilayers. This is done by exchanging the lipid filmon the sub phase between depositions.

Another way of preparing supported bilayers is the vesicle fusion method(A. A. Brian and H. M. McConnell. Allogenic stimulation of cytotoxic Tcells by supported planar membranes. Proc. Natl. Acad. Sci. USA,81:6159-6163, 1984). A solution of small unilamellar vesicles (SUVs) isapplied onto the surface of a piece of hydrophilized silicon or freshlycleaved mica. When this sample is left at low temperature (4° C.) thevesicles fuse with the surface to make a continuous bilayer. Withoutbeing bound to any theory it has been hypothesized that the vesiclesfirst adsorb to the surface of the substrate then fuse to make a flat,pancake-like structure and finally rupture and spread out resulting in asingle bilayer on the surface (I. Reviakine and A. Brisson. Formation ofsupported phospholipid bilayers from unilamellar vesicles investigatedby atomic force microscopy. Langmuir, 16:1806-1815, 2000). It has alsobeen suggested that after fusion with the substrate only the part of thevesicle which is in direct contact with the substrate becomes thesupported bilayer (Z. V. Leonenko, A. Carnini, and D. T. Cramb.Supported planar bilayer formation by vesicle fusion: the interaction ofphospholipid vesicles with surfaces and the effect of gramicidin onbilayer properties using atomic force microscopy. Biochim. Biophys.Acta, 1509:131-147, 2000). With this mechanism the vesicle ruptures atthe edges with the highest curvature and the top part of the bilayer maythen migrate to the surface of the substrate to increase the size of theformed supported bilayer. It has been reported that bilayers are formedwithin minutes of applying the solution onto the substrate but thisshort incubation time may result in incomplete bilayers. Hours orovernight incubation have also been reported (E. Reimhult, F. Hook, andB. Kasemo. Intact vesicle adsorption and supported biomembrane formationfrom vesicles in solution: Influence of surface chemistry, vesicle size,temperature, and osmotic pressure. Langmuir, 19:1681-1691, 2003; H. A.Rinia, R. A. Kik, R. A. Demel, M. M. E. Snel, J. A. Killian, J. P. J. M.van der Eerden, and B. de Kruijff. Visualization of highly orderedstriated domains induced by transmembrane peptides in supportedphosphatidylcholine bilayers. Biochemistry, 39:5852-5858, 2000).

A third technique which can be used to prepare supported bilayers isspin-coating (E. Reimhult, F. Hook, and B. Kasemo. Intact vesicleadsorption and supported biomembrane formation from vesicles insolution: Influence of surface chemistry, vesicle size, temperature, andosmotic pressure. Langmuir, 19:1681-1691, 2003; A. C. Simonsen and L. A.Bagatolli. Structure of spin-coated lipid films and domain formation insupported membranes formed by hydration. Langmuir, 20:9720-9728, 2004).In spin-coating the lipid is dissolved in a suitable solvent and adroplet is placed on the substrate which is then rotated while thesolvent evaporates and a lipid coating is produced. Depending on theconcentration of the lipid solution the spin-coated film consist of oneor more lipid bilayers. However, upon hydration the multiple layers havebeen shown to be unstable, and usually only one supported bilayerremains on the surface. This procedure is easy and fast and it has beenexercised with low-melting lipids (POPC) as well as lipids withintermediate (DPPC) and very high transition temperature (ceramide).Useful lipids include, e.g., phospholipids and amphiphilic lipids.

In some embodiments, peptides and proteins are incorporated in thesupported bilayers by using the vesicle fusion technique. Many membraneproteins may denature in organic solvents especially if they containlarge domains exposed to the aqueous solution on either side of themembrane. In some embodiments, it is therefore preferred to insert thepeptides or proteins in vesicles. Many peptides and proteins (e.g.aquaporins and hydrophobin) can be co-solubilized with lipid in theorganic solvent prior to formation of vesicles and the peptidecontaining vesicles are then applied to the substrate. This has beendone with a number of peptides, for example WALP (H. A. Rinia, R. A.Kik, R. A. Demel, M. M. E. Snel, J. A. Killian, J. P. J. M. van derEerden, and B. de Kruijff. Visualization of highly ordered striateddomains induced by transmembrane peptides in supportedphosphatidylcholine bilayers. Biochemistry, 39:5852-5858, 2000),gramicidin (J. Mou, D. M. Czajkowsky, and Z. Shao. Gramicidin Aaggregation in supported gel state phosphatidylcholine bilayers.Biochemistry, 35:3222-3226, 1996), clavanin A (E. J. M. van Kan, D. N.Ganchev, M. M. E. Snel, V. Chupin, A. van der Bent, and B. de Kruijff.The peptide entibiotic clavanin A interacts strongly and specificallywith lipid bilayers. Biochemistry, 42:11366-11372, 2003) and Amyloid βProtein (H. Lin, R. Bhatia, and R. Lal. Amyloid beta-protein forms ionchannels: implications for Alzheimer's disease pathophysiology. FASEBJ., 15:2433-2444, 2001). In some embodiments, membrane proteins such asaquaporins are inserted into vesicles by other means. This can be doneusing the strategies for reconstitution of membrane proteins intovesicles as described for cytochrome c oxidase as a model protein in theintroduction to chapter 4 on pages 41-45 of the herein incorporatedthesis “Supported bilayers as models of biological membranes” byDanielle Keller, February 2005, MEMPHYS-center for biomembrane physics,Physics Department, University of Southern Denmark and Danish PolymerCentre, Riso National Laboratory, Denmark.

Multi layer stacking of the individual 2D-arrays are possible and may bedesirable. The final dimensions of the stacked arrays will depend onoverall robustness and on intrinsic permeability of the chosen membranematerial/membrane composition. Stacking might depart from a system whereproteins trivially are embedded in a single, probably supported, lipidbilayer. A subsequent series of collapsing vesicles events on thesupported bilayer could then provide multi layer devices, given that thevesicles prerequisite are reconstituted with an appropriatepolypeptide(s) (e.g. aquaporin and/or hydrophobin). Incorporation of thestacked unit-device into a stabilizing membrane or stabilizing polymermatrix and subsequent stitching of these individual units would yield anoverall mesh, eventually via self-assembly processes.

In some embodiments, the biomimetic membranes comprise block copolymermembranes simulating a natural environment. In some embodiments, thepolypeptide(s) described herein (e.g. aquaporin and/or hydrophobin) areincorporated into the block copolymer membranes simulating a naturalenvironment.

In some embodiments, a method of forming a biocompatible membrane foruse with block copolymer-based membrane, is as follows: form a solutionof block copolymer in solvent (BC solution). The solution can be amixture of two or more block copolymers. The solution contains 1 to 90%w/v copolymer, or 2 to 20%, or 5 to 10%, such as 7%. Prepare apolypeptide (e.g. aquaporin) solution in the prepared BC solution,preferably by adding 1.0 to 50.0 mg/mL of the selected polypeptide, or1.0 to 10.0 mg/mL. Drop a small volume (e.g., 4 microliter)polypeptide/BC solution onto each aperture or each of a subset ofapertures, and allow to dry, thereby removing the solvent. Repeat thisstep as needed to cover all apertures.

The solvent is selected to be miscible with both the water componentused in the process and the B component of the block copolymer.Appropriate solvents are believed to include methanol, ethanol,2-propanol, 1-propanol, tetrahydrofuran, 1,4-dioxane, solvent mixturesthat can include more apolar solvents such as dichloromethane so long asthe mixture has the appropriate miscibility, and the like. (Solventcomponents that have any tendency to form protein-destructivecontaminants such as peroxides can be appropriately purified andhandled.) Solvent typically comprises 100% v/v or more of the appliedpolypeptide/BC solution, preferably 20% or more, and usefully 30% ormore.

The above-described method of introducing a polypeptide(s) to a solutioncontaining non-aqueous solvent(s) in the presence of block copolymersserves to stabilize the function of active polypeptides. The non-aqueouscomponents can comprise all of the solvent.

In some embodiments, the biomimetic membrane is composed of one or morevesicles having hydrophilic inner and outer layers and a middlehydrophobic layer. In some embodiments the one or more vesicles arecomposed of lipids, polymers and/or a combination thereof. In someembodiments, the vesicles can be crosslinked to provide additionalstability. It should be understood that, when copolymers are used, thecopolymers can be polymerized via end groups, crosslinked via internalcrosslinkable groups, or a combination of end group and internal grouppolymerization/crosslinking can be used. In some embodiments, theinvention provides lipid and/or copolymer vesicles supported by asubstrate (e.g. microporous substrate). In some embodiments, thesupported lipid and/or copolymer vesicles are coated with one or morehydrophobins as described herein.

The biomimetic membranes used in the invention can be prepared accordingto the teachings in PCT/DK2006/000278, U.S. Pat. No. 8,123,948, USpublication number 2004/0049230, US publication number 2011/0020950, USpublication number 2011/0046074, WO 2012/161662, and PCT/SG2012/000063.

In some embodiments, the biomimetic membrane may comprise a sandwichconstruction having at least two permeable support layers separated byat least one lipid bilayer or block copolymer comprising one or morefunctional polypepeptides (e.g. aquaporin water channels and/orhydrophobin). In some embodiments, the biomimetic membrane may comprisea sandwich construction having at least two permeable support layersseparated by at least one or more vesicles (e.g. lipid or polymer)comprising one or more functional polypepeptides (e.g. aquaporin waterchannels and/or hydrophobin).

In some embodiments, the biomimetic membrane may consist of anamphiphilic biomimetic membrane, such as a membrane comprising lipidsdescribed herein, or a block copolymer. In some embodiments, the lipidbilayer(s) essentially consist(s) of amphiphilic lipids selected fromthe group consisting of phospholipids, phosphoglycerides, sphingolipids,and cardiolipin, as well as mixtures thereof, e.g. phospholipids such as1,2-dipalmitoyl-sn-phosphatidylcholine (DPPC), or mixtures ofphospholipids. Alternatively, the lipid bilayers may consist essentiallyof or contain polymerizable lipids.

In some embodiments, the biomimetic membrane may consist of polypeptidesbased thin film composite membranes. Lipid or polymer vesiclescontaining the functional polypeptides of the invention (e.g. aquaporinwater channels and/or hydrophobin) are incorporated in an ultrathinselective layer, and are supported by a substrate (e.g. a microporoussubstrate). In some embodiments, thin film composite membrane is createdon a surface of a porous substrate by interfacial polymerization of anaqueous solution of an (aromatic) amine having an added suspension ofamphiphilic lipid/copolymer vesicles followed by addition of an acidchloride solution in an organic solvent to allow the amine and the acylchloride to form a polyamide active TFC layer wherein. During theformation of thin polyamide film, the vesicles, which may be in the formof liposomes or polymersomes with or without incorporated protein(proteoliposomes or proteopolymersomes), become part of the activelayer.

In some embodiments, the biomimetic membrane comprises reconstitutedpolypeptides (e.g. aquaporin water channels and/or hydrophobin) in lipidbilayers in contact with a porous support. Any suitable support layerthat is compatible with the preparation of the biomimetic membranes isencompassed in the methods, compositions and apparatuses of theinvention. In some embodiments, aquaporin based thin film compositemembranes are provided. Amphiphilic lipid-AQP/HFBII and/or amphiphiliccopolymer-AQP/HFBII vesicles can be incorporated with ultrathinselective layer, and then can be supported by a microporous substrate.

Examples of useful support materials with a hydrophilic surface for thepreparation of biomimetic membranes according to the invention includebut are not limited to, mica such as muscovite, mica tape, polysulfon,AlO₂, and polymeric materials having a hydrophilic surface, e.g.cellulose. The support materials are essentially planar which means thatthe support is preferably planar, but curvature of the support isallowable, such as needed when spirally wound membranes aremanufactured. In this case the support material is preferably flexible,such as cellulose membranes.

In some embodiments, the porous support may comprise a material such asmica having an essentially planar structure with a hydrophilic surfaceand wherein micro or nano pores have been formed, e.g. by etching.Hence, in some embodiments, the permeable support layer comprises anessentially planar, hydrophilic layer comprising mica or mica tapehaving a layer thickness in the mm to μm scale and wherein nanoporeshaving a diameter of less than approximately 50 nm (typically in the10-40 nm range) have been formed (e.g. by etching such as by atrack-etch technique). The mica is preferably muscovite.

The permeable support layers may also comprise a hydrophilized membranesurface, such as a membrane selected from the group consisting ofsilicone membranes, polysulfon, AlO₂, and polymers such as cellulosehaving a hydrophilic surface, wherein nanopores having a diameter ofless than approximately 50 nm (typically in the 10-40 nm range) havebeen formed.

The biomimetic membrane comprising the polypeptide(s) described herein(e.g., aquaporin channels and/or hydrophobin) may be a bilayerresembling the natural constitution of biological cell membranes, or thebiomimetic membrane may consist of multiple bilayers of fused depositedlipid vesicles. In some embodiments, the lipids are of amphiphilicnature, such as the phospholipids (or phosphoglycerides), sphingolipidsand cardiolipin. When depositing the lipid layers on the poroussubstrate, the polypeptide(s) may preferably be deposited adjacent to orin the preexisting pores in the support material.

In some embodiments, the permeable or porous support used is preparedaccording to R. M. Webber, J. L. Anderson, M. S. John, Macromolecules 23(1990), 1026-1034.

In some embodiments, the inventions provides for the reconstitution ofpolypeptide(s) (e.g., aquaporin water channels and/or hydrophobin) in aplanar lipid bilayer assembled around a porous support membrane with ahydrophobic surface, such as Teflon® film, where lipid monolayersassemble on each side of the porous support membrane. In the pores ofthe porous support membrane lipid bilayers will assemble, where thepolypeptide(s) can be reconstituted.

In some embodiments, the invention provides a biomimetic membranecomprising a sandwich construction having at least two lipid monolayers,which, when assembled into one bilayer, comprises functionalpolypeptides, said at least two lipid monolayers being separated by atleast one permeable support layer. In some embodiments, the supportlayer comprises a hydrophobic perforated material which forms thecontact surface with the lipid monolayers and wherein the lipid bilayeris formed in the perforations of the hydrophobic perforated material.

In some embodiments, the invention provides a biomimetic membranecomprising lipid and/or polymer, vesicles, comprising the functionalpolypeptides of the invention (e.g. aquaporin water channels and/orhydrophobin) supported in a substrate. In some embodiments, theinvention provides a biomimetic membrane comprising lipid and/or polymervesicles, comprising the functional polypeptides of the invention,incorporated into an ultrathin selective layer, and supported by asubstrate a microporous substrate.

In some embodiments, the hydrophobic material has a degree ofhydrophobicity corresponding to a contact angle of at least 100° C.between a droplet of deionized water and the hydrophobic material, wherethe contact angle measurement is performed at 20° C. and atmosphericpressure, but higher degrees of hydrophobicity are preferred, such asthose corresponding to contact angles of at least 105° C., 110° C., 120°C. and 120° C. In some embodiments. The hydrophobic materials areparafilm or Teflon®.

The hydrophobic material is typically planar (but may be flexible andthus curved) and the perforations are typically evenly distributed andsubstantially all of substantially the same geometric shape in theintermediate plane between the 2 surfaces of the hydrophobic material.

The “intermediate plane” is defined as the plane consisting of pointsfrom which the perpendicular distance to either both of the 2 surfacesof the planar hydrophobic material is the same.

The size of the perforations in the hydrophobic material should merelyensure that stable membranes (e.g., lipid and/or polymers; monolayers orbilayers of amphiphilic lipids and/or polymers) can be formed in theperforations, so they may have sizes in the nm, μm or mm range. Themembranes can be in the shape of vesicles (e.g. lipid and/or polymers).

In some embodiments, the hydrophobic material is perforated in such away that the ratio between perforation are and non-perforated area ofthe material is maximized, since this provides a maximum area of lipidbilayer with polypeptides that form channels (e.g. aquaporins) to effectwater transport. The pattern constituted by the perforations is thus ofimportance as is the distance between each perforation. An optimumpattern is a hexagonal arrangement of the perforations with a minimum“wall thickness” between each perforation in the pattern. However, atquadratic pattern may also prove sufficient.

In some embodiments, the biomimetic membrane used comprises anamphiphilic biomimetic membrane, such as a membrane comprising lipidsdescribed herein. Thus, the lipid bilayer(s) essentially consist(s) ofamphiphilic lipids selected from the group consisting of phospholipids,phosphoglycerides, sphingolipids, and cardiolipin, as well as mixturesthereof, e.g. phospholipids such as1,2-dipalmitoyl-sn-phosphatidylcholine (DPPC), or mixtures ofphospholipids. The difference from the first aspect is primarily thatthe membrane only constitutes a bilayer in areas where the hydrophobicsupport is perforated, whereas the lipids are organized with theirhydrophobic ends facing the hydrophobic support and the hydrophilic endsfacing the aqueous environment.

In some embodiments, the biomimetic membranes comprise one or moreaquaporin. Examples of aquaporins that can be used for the preparationof biomimetic membranes according to the invention included, but are notlimited to: AQP1, TIP, PIP, NIP, and mixtures and hybrids thereof. Theaquaporins of plant origin are especially desirable, since the risk ofincluding contaminants, such as pathogenic viruses and prions, which canbe harmful to humans is greatly reduced. In addition, the plantaquaporins are natural gene products of plants and can be overexpressedand produced in plants. In some embodiments, the aquaporin water channelis aquaporin Z (AqpZ), which is derived from E. Coli. In someembodiments, the aquaporin channels are modified.

In some embodiments, the aquaporin water channel is selected from thegroup consisting of aquaglyceroporins (GLpF), such as a GLPA channel, aGLPB1 channel, a GLPB2 channel, a GLPB3 channel, and a GLPY2 channel,and mixtures and hybrids thereof.

In some embodiments, the biomimetic membranes further comprise or morehydrophobins in combination with one or more aquaporins. In someembodiments, the hydrophobin is selected from the group consisting of:(a) HFBII (SEQ ID NO: 2; obtainable from the fungus Trichoderma reesei);(b) HFBI (SEQ ID NO: 4; obtainable from the fungus Trichoderma reesei);(c) SC3 (SEQ ID NO: 6; obtainable from the fungus Schizophyllumcommune); (d) EAS (SEQ ID NO: 8; obtainable from the fungus Neurosporacrassa); and (e) TT1 (SEQ ID NO: 10; obtainable from the fungusTalaromyces thermophilus); or a protein having at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, or at least 99% sequence identity in the hydrophobin core to anythereof.

In some embodiments, the hydrophobin is “HFBII” (SEQ ID NO: 2;obtainable from Trichoderma reesei) or a protein having at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, or at least 99% sequence identity in the hydrophobin corethereof.

In some embodiments, the biomimetic membranes used in the invention areenclosed in a stabilizing permeable or porous membrane which may berigid or flexible and which may serve as protection of the watermembrane as well as a pre-filter to exclude coarse particulate matterfrom the aqueous liquid to be purified. Alternatively or additionally,the biomimetic membrane of the invention may be deposited on a filterdisk to create a water filter.

Biomimetic membranes can be formed against a solid material, such as bycoating onto glass, carbon that is surface modified to increasehydrophobicity, or a polymer (such as polyvinyl acetate, PDMS, Kapton®,a perfluorinated polymer, Teflon®, PVDF, PEEK, polyester, or UHMWPE,polypropylene or polysulfone). Polymers such as PDMS provide anexcellent support that can be used to establish openings on whichbiocompatible membranes can be formed.

Useful porous materials for the preparation of biomimetic membranesaccording to the Mueller based lipid bilayer membranes (Mueller, D.,Rudin, O., Tien, H. T. and W. C. Wescott. Reconstruction of cellmembrane structure in virto and its transformation into an excitablesystem. Nature (Lond.) 194: 979-980, 1962) or the Montal decane basedmembranes (Montal, M., and P. Mueller. Formation of bimolecularmembranes from monolayers and study of their properties. Proc. Natl.Acad. Sci. USA. 69:3561-3566, 1972) are, Teflon® films and other porousmembrane materials with hydrophobic surface properties.

The invention also relates to the upscaling of the biomimetic membranes,where multiple holes are formed in a Teflon® partition film or anothermaterial with hydrophobic surface properties, and a lipid bilayermembrane or a block copolymer membrane comprising the polypeptide(s) areformed around the material.

Useful materials for the stabilizing membrane optionally used to enclosethe biomimetic membranes described herein are micro-porous siliconemembranes having a relatively small pore size and which solidifies atabout room temperature or at a temperature below about 50° C.

c. Linking Carriers

In some embodiments, the biomimetic membranes comprise a linkingcarrier. The term linking carrier includes entities that serve to linkagents, e.g., targeting agents, to the biomimetic membranes. In someembodiments, the linking carrier confers additional advantageousproperties to the biomimetic membranes. Examples of these additionaladvantages include, but are not limited to: 1) multivalency, which isdefined as the ability to attach multiple agents to the biomimeticmembranes; and 2) improved stability.

In some embodiments, the linking carriers are biocompatible polymers(such as dextran) or macromolecular assemblies of biocompatiblecomponents. Examples of linking carriers include, but are not limitedto, microbubbles, polymerized microbubbles, micelles, liposomes, otherlipid vesicles, dendrimers, polyethylene glycol assemblies, cappedpolylysines, poly(hydroxybutyric acid), dextrans, and coated polymers.

The linking carrier can be coupled to an agent (e.g. stabilizing agent)by a variety of methods, depending on the specific chemistry involved.The coupling can be covalent or non-covalent. A variety of methodssuitable for coupling of the targeting entity and the therapeutic entityto the linking carrier can be found in Hermanson, “BioconjugateTechniques”, Academic Press: New York, 1996; and in “Chemistry ofProtein Conjugation and Cross-linking” by S. S. Wong, CRC Press, 1993.Specific coupling methods include, but are not limited to, the use ofbifunctional linkers, carbodiimide condensation, disulfide bondformation, and use of a specific binding pair where one member of thepair is on the linking carrier and another member of the pair is on thetherapeutic or targeting entity, e.g. a biotin-avidin interaction.

d. Other Stabilizing Entities

In some embodiments, the biomimetic membranes contain a stabilizingentity. As used herein, “stabilizing” refers to the ability to impartadditional advantages to the biomimetic membranes, for example, physicalstability, e.g., longer half-life, colloidal stability, and/or capacityfor multivalency; that is, increased payload capacity due to numeroussites for attachment of targeting agents. Stabilizing entities includemacromolecules or polymers, which may optionally contain chemicalfunctionality for the association of the stabilizing entity to thesurface of the biomimetic membrane, and/or for subsequent association oftherapeutic agents and/or targeting agents. The polymer should bebiocompatible with aqueous solutions. Polymers useful to stabilize thebiomimetic membranes of the present invention may be of natural,semi-synthetic (modified natural) or synthetic origin. A number ofstabilizing entities which may be employed in the present invention areavailable, including xanthan gum, acacia, agar, agarose, alginic acid,alginate, sodium alginate, carrageenan, gelatin, guar gum, tragacanth,locust bean, bassorin, karaya, gum arabic, pectin, casein, bentonite,unpurified bentonite, purified bentonite, bentonite magma, and colloidalbentonite.

Other natural polymers include naturally occurring polysaccharides, suchas, for example, arabinans, fructans, fucans, galactans, galacturonans,glucans, mannans, xylans (such as, for example, inulin), levan,fucoidan, carrageenan, galatocarolose, pectic acid, pectins, includingamylose, pullulan, glycogen, amylopectin, cellulose, dextran, dextrose,dextrin, glucose, polyglucose, polydextrose, pustulan, chitin, agarose,keratin, chondroitin, dermatan, hyaluronic acid, alginic acid, xanthingum, starch and various other natural homopolyner or heteropolymers,such as those containing one or more of the following aldoses, ketoses,acids or amines: erythrose, threose, ribose, arabinose, xylose, lyxose,allose, altrose, glucose, dextrose, mannose, gulose, idose, galactose,talose, erythrulose, ribulose, xylulose, psicose, fructose, sorbose,tagatose, mannitol, sorbitol, lactose, sucrose, trehalose, maltose,cellobiose, glycine, serine, threonine, cysteine, tyrosine, asparagine,glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine,glucuronic acid, gluconic acid, glucaric acid, galacturonic acid,mannuronic acid, glucosamine, galactosamine, and neuraminic acid, andnaturally occurring derivatives thereof. Other suitable polymers includeproteins, such as albumin, polyalginates, and polylactide-glycolidecopolymers, cellulose, cellulose (microcrystalline), methylcellulose,hydroxyethylcellulose, hydroxypropylcellulose,hydroxypropylmethylcellulose, carboxymethylcellulose, and calciumcarboxymethylcellulose.

Exemplary semi-synthetic polymers include carboxymethylcellulose, sodiumcarboxymethylcellulose, carboxymethylcellulose sodium 12,hydroxymethylcellulose, hydroxypropylmethylcellulose, methylcellulose,and methoxycellulose. Other semi-synthetic polymers suitable for use inthe present invention include carboxydextran, aminodextran, dextranaldehyde, chitosan, and carboxymethyl chitosan.

Exemplary synthetic polymers include poly(ethylene imine) andderivatives, polyphosphazenes, hydroxyapatites, fluoroapatite polymers,polyethylenes (such as, for example, polyethylene glycol, the class ofcompounds referred to as Pluronics®, commercially available from BASF,(Parsippany, N.J.), polyoxyethylene, and polyethylene terephthlate),polypropylenes (such as, for example, polypropylene glycol),polyurethanes (such as, for example, polyvinyl alcohol (PVA), polyvinylchloride and polyvinylpyrrolidone), polyamides including nylon,polystyrene, polylactic acids, fluorinated hydrocarbon polymers,fluorinated carbon polymers (such as, for example,polytetrafluoroethylene), acrylate, methacrylate, andpolymethylmethacrylate, and derivatives thereof, polysorbate, carbomer934P, magnesium aluminum silicate, aluminum monostearate, polyethyleneoxide, polyvinylalcohol, povidone, polyethylene glycol, and propyleneglycol. Methods for the preparation of biomimetic membranes which employpolymers to stabilize biomimetic membranes compositions will be readilyapparent to one skilled in the art, in view of the present disclosure,when coupled with information known in the art, such as that describedand referred to in Unger, U.S. Pat. No. 5,205,290, the disclosure ofwhich is hereby incorporated by reference herein in its entirety.

In some embodiments, the stabilizing entity is dextran. In someembodiments, the stabilizing entity is a modified dextran, such as aminodextran. In some embodiments, the stabilizing entity is poly(ethyleneimine) (PEI).

In some embodiments, the following polymers and their derivatives areused: poly(galacturonic acid), poly(L-glutamic acid), poly(L-glutamicacid-L-tyrosine), poly[R)-3-hydroxybutyric acid], poly(inosinic acidpotassium salt), poly(L-lysine), poly(acrylic acid),poly(ethanolsulfonic acid sodium salt), poly(methylhydrosiloxane),polyvinyl alcohol), poly(vinylpolypyrrolidone), poly(vinylpyrrolidone),poly(glycolide), poly(lactide), poly(lactide-co-glycolide), andhyaluronic acid. In some embodiments, copolymers including a monomerhaving at least one reactive site, or multiple reactive sites, are usedfor the attachment of the copolymer to the biomimetic membrane or othermolecules.

In some embodiments, the polymer may act as a hetero- orhomobifunctional linking agent for the attachment of agents, proteins orchelators such as DTPA and its derivatives.

In some embodiments, the stabilizing entity is associated with thebiomimetic membranes by covalent means. In another embodiment, thestabilizing entity is associated with the biomimetic membranes bynon-covalent means. Covalent means for attaching the targeting entitywith the biomimetic membranes are known in the art and described in theUS publication 2010/0111840, incorporated by reference herein in itsentirety.

Noncovalent means for attaching agents to the biomimetic membranesinclude but are not limited to attachment via ionic, hydrogen-bondinginteractions, including those mediated by water molecules or othersolvents, hydrophobic interactions, or any combination of these.

In some embodiments, the stabilizing agent forms a coating on thebiomimetic membrane.

In some embodiments, the biomimetic membranes of the invention may alsobe linked to functional agents, such as poly(ethylene glycol) (PEG),that otherwise modify the biomimetic membrane properties, such asaggregation tendencies, binding by opsonizing proteins, uptake by cells,and stability.

e. Uses of Membranes

In some embodiments, the biomimetic membranes comprise functionalmolecules, such as channel forming molecules, e.g. certain peptides orpeptide like molecules including amphotericin B, alamethicin,valinomycin, gramicidin A and their dimers, oligomers and analoguesthereof; or transmembrane proteins, e.g. aquaporin water channels, Fasprotein, DsbB, CFTR, alpha-haemolysin, VDAC, and OmpG.

In some embodiments, the present invention relates to biomimeticmembranes comprising functional channel forming molecules. In someembodiments, the channel-forming molecule is selected among theaquaporin water channels to make it possible to obtain a biomimeticmembrane useful in a filtration device for purification of a watersource or a liquid, aqueous medium. In some embodiments, the biomimeticmembranes further comprises one or more hydrophobins. Other usefulapplications include a biosensor or for high throughput screening ofligands.

In some embodiments, the invention provides membrane scaffold containingapertures and the formation of bilayer biomimetic membranes in saidapertures

In some embodiments, the biomimetic membranes of the invention aresuitable for incorporation of biomolecules that are naturallymembrane-bound, e.g. aquaporins, or for incorporation of artificialmolecules. The biomimetic membranes comprising aquaporins are suitablefor transporting water from one side of the membrane to the other side,e.g. when driven by a pressure gradient. The ability to transport watermay be utilized in a filtration device for preparing essentially purewater. This facilitates processes such as water purification,desalinization, and molecular concentration through dialysis. Otherembodiments of the biomimetic membranes are suitable as biosensors orfor high throughput screening of transmembrane protein ligands. In someembodiments, the channel-forming molecules cover at least 1% of themembrane surface. In some embodiments, the membrane is covered with 1 to10% of the channel-forming molecules.

In some embodiments, the invention relates in a further aspect to afiltration device for filtering essentially pure water comprising abiomimetic membrane comprising aquaporin water channels and one or morehydrophobins, as described above. The advantages of using the biomimeticmembrane in said filtering device or other applications where upscale isan advantage is closely related to the possibility of up-scaling thefunctional membrane area by the manufacturing of large, flexible, andrelatively thin sheets having a large multitude of discrete membraneunits. In addition, the biomimetic membrane ensures that filteringability is maintained even though one or more discrete membrane unitshave failed. This situation may especially apply to a filtration devicehaving multi layer stacking of the individual biomimetic membranes or2D-aperture-arrays.

In some embodiments, the present invention provides biomimetic membranescomprising one or more polypeptides (e.g. aquaporins and/or hydrophobin)used for pressure retarded osmosis. In some embodiments, the presentinvention provides the implementation of said membrane in apressure-retarded osmosis (PRO) system used in the production ofsalinity power. In some embodiments, the present invention providesbiomimetic membranes comprising aquaporins and/or hydrophobin used forPRO. The lipids membranes comprising one or more polypeptides (e.g.aquaporins and/or hydrophobin) can be produced using multiple differentprocedures known in the art, including those described herein.

In some embodiments, the invention includes the use of the membrane toextract excess water from aqueous substances or solutions, e.g. toobtain increased concentration of a desirable solute.

In some embodiments, proteins in the biomimetic membrane can be used totransport electrons/protons to enable the transduction of electrical andchemical power, and act as mechanical valves and sensors.

In some embodiments, the biomimetic membrane is used to provide abiosolar-powered material which comprise of a biomimetic membraneembedded with one or more energy converting proteins, e.g.,bacteriorhodopsin and cytochrome oxydase, which will convert opticalenergy to electrical energy and deliver this energy to an external load.In some embodiments, the biosolar-powered material further comprises afabric which consists of a thin fabric incorporating the lipid polymermembrane embedded with the one or more energy converting proteins asdescribed in US publication no. 2004/0049230.

Other useful applications of said biomimetic membranes include biosensorapplications, such as a transmembrane protein functioning as receptor orchannel, labeled with a fluorophore to make a protein-based biosensorsensitive to ligands, solutes or small molecules. Said biosensorsincorporated into biomimetic membranes can be used for ligand-receptorinteractions used in high throughput screening assays for diagnostic orprognostic purposes prepared in 96-multi well plates, lab-on-a-chipdevices or build into point-of-care measuring devices, or serve asquantitative measuring devices of solutes or small molecules such asheavy metal ions e.g. cadmium, copper, lead, etc., or antibiotics andother polluting agents for quantitative on-the-spot water analysis, orblood analysis of animals and humans.

In some embodiments of the present invention involves the use ofbiomimetic membranes for the classification, diagnosis, prognosis of acondition, determination of a condition stage, determination of responseto treatment, monitoring and predicting outcome of a condition. Anotherembodiment of the invention involves the use of the biomimetic membranesdescribed herein in monitoring and predicting outcome of a condition.Another embodiment of the invention involves the use of the biomimeticmembranes described herein in drug screening, to determine which drugsmay be useful in particular diseases. Another embodiment of theinvention involves the use of the biomimetic membranes described hereinfor the treatment of a condition.

In some embodiments, the methods involve the administration of one ormore biomimetic membranes for the treatment of one or more conditions.Combinations of agents can be used to treat one condition or multipleconditions or to modulate the side-effects of one or more agents in thecombination.

The term “treating” and its grammatical equivalents as used hereinincludes achieving a therapeutic benefit and/or a prophylactic benefit.By therapeutic benefit is meant eradication or amelioration of theunderlying condition being treated. Also, a therapeutic benefit isachieved with the eradication or amelioration of one or more of thephysiological symptoms associated with the underlying condition suchthat an improvement is observed in the patient, notwithstanding that thepatient may still be afflicted with the underlying condition. Forprophylactic benefit, the compositions may be administered to a patientat risk of developing a particular disease, or to a patient reportingone or more of the physiological symptoms of a disease, even though adiagnosis of this disease may not have been made.

As used herein the term “diagnose” or “diagnosis” of a conditionincludes predicting or diagnosing the condition, determiningpredisposition to the condition, monitoring treatment of the condition,diagnosing a therapeutic response of the disease, and prognosis of thecondition, condition progression, and response to particular treatmentof the condition.

Methods

In some embodiments, the present invention provides methods forstabilizing a membrane protein, a membrane, and/or a membrane comprisinga membrane protein using one or more hydrophobins. In some embodiments,the present invention provides methods for stabilizing a biomimeticmembrane using one or more hydrophobins. In some embodiments, thepresent invention provides methods for stabilizing membrane proteinusing one or more hydrophobins. In some embodiments, the presentinvention provides methods for stabilizing a biomimetic membranecontaining a membrane protein using one or more hydrophobins.

In some embodiments, the methods of the invention comprise adding one ormore hydrophobins to a membrane protein to stabilize the membraneprotein. In some embodiments, the membrane protein is an isolated and/orpurified membrane protein. In some embodiments, the one or morehydrophobins are added to the membrane protein in an amount sufficientto increase the stability of the membrane protein. In some embodiments,the one or more hydrophobins are added to the membrane protein in anamount sufficient to increase the thermal stability of the membraneprotein.

In some embodiments, the methods of the invention for stabilizing amembrane protein comprise adding one or more hydrophobins to a membraneprotein to disperse the membrane protein. Thus, in some embodiments, themembrane protein molecules are held in dispersion in a medium. In someembodiments, the membrane protein is an isolated and/or purifiedmembrane protein. In some embodiments, the one or more hydrophobins areadded to the membrane protein in an amount sufficient to disperse themembrane protein. The invention provides stable dispersions of themembrane proteins. The dispersed membrane protein can then be used inthe formation of biomimetic membranes.

In some embodiments, the methods of the invention comprise adding one ormore hydrophobins to a biomimetic membrane to stabilize the biomimeticmembrane. In some embodiments, the one or more hydrophobins are added tothe biomimetic membrane in an amount sufficient to increase thestability of the biomimetic membrane protein. In some embodiments, theone or more hydrophobins are added to the biomimetic membrane in anamount sufficient to increase the thermal stability of the biomimeticmembrane.

In some embodiments, the methods of the invention comprise adding one ormore hydrophobins to a biomimetic membrane containing a membrane proteinto stabilize the biomimetic membrane and/or the membrane protein. Insome embodiments, the one or more hydrophobins are added to thebiomimetic membrane containing a membrane protein in an amountsufficient to increase the stability of the biomimetic membrane proteinand/or the membrane protein. In some embodiments, the one or morehydrophobins are added to the biomimetic membrane containing themembrane protein in an amount sufficient to increase the thermalstability of the biomimetic membrane and/or the membrane protein. Insome embodiments, the hydrophobin concentration is between 0.01 mm-1 M.In some embodiments, the hydrophobin concentration is between 0.01mM-0.05 mM. In some embodiments, the hydrophobin concentration isbetween 0.01 mM-20 mM. In some embodiments, the hydrophobinconcentration is between 0.1 mM-0.5 M. In some embodiments, thehydrophobin concentration is between 0.1 mM-200 mM. In some embodiments,the hydrophobin concentration is between 0.1 mM-100 mM. In someembodiments, the hydrophobin concentration is between 0.1 mM-100 mM. Insome embodiments, the hydrophobin concentration is between 0.1 mM-50 mM.In some embodiments, the hydrophobin concentration is between 0.1 mM-20mM. In some embodiments, the hydrophobin concentration is between 0.1mM-10 mM. In some embodiments, the hydrophobin concentration is between0.1 mM-5 mM. In some embodiments, the hydrophobin concentration isbetween 0.1 mM-1 mM. In some embodiments, the hydrophobin is present ina concentration of 0.1 μM-50 mM. In some embodiments, the hydrophobin ispresent in a concentration of 0.1-20% by weight of the total weight ofthe composition.

In some embodiments, the methods of the invention for stabilizing abiomimetic membrane comprise adding one or more hydrophobins as anemulsifier to facilitate the biomimetic membrane formation. In someembodiments, the biomimetic membrane is a lipid or polymer vesicle (e.g.liposome or polymerosome). In some embodiments, the methods of theinvention for stabilizing a biomimetic membrane comprise adding one ormore hydrophobins as an emulsifier to facilitate lipid/polymer vesicleformation and to modulate the lipid/polymer vesicle size distribution.

In some embodiments, the methods of the invention comprise adding one ormore hydrophobins to disperse a membrane protein, to emulsifylipid/polymer vesicle (e.g. liposome or polymerosome) formation, and toform a continuous coating on said lipid/polymer vesicles.

In some embodiments, the methods of stabilizing a membrane proteincomprise the following steps: (a) providing an isolated membraneprotein; (b) adding the isolated membrane protein to an aqueoussolution; and (c) adding one or more hydrophobins as described herein.The steps (b) and (c) in the method above can be performedsimultaneously or sequentially.

In some embodiments, the methods comprise the following steps: (a)providing an isolated membrane protein and (b) drying the membraneprotein on a surface in the presence of one or more hydrophobins.

The surface can be any surface that does not react with the membraneprotein and/or the one or more hydrophobins. Exemplary surfaces include,but are not limited to, glass slides, plastic slides, a multi-well plateand the like. As used herein, drying is meant to include the removal ofwater or dehydration of the membrane protein. Drying can be accomplishedby methods well known in the art such as lyophilization and/or flashfreezing in liquid nitrogen.

In some embodiments, the methods of stabilizing a biomimetic membranecomprise the following steps: (a) providing the biomimetic membrane; and(b) adding one or more hydrophobins as described herein to thebiomimetic membrane.

In some embodiments, the methods of stabilizing a biomimetic membranecomprise the following steps: (a) providing the components (e.g. lipidand/or polymers) for the formation of the biomimetic membrane, (b)adding one or more hydrophobins as described herein to the components,and (c) forming the biomimetic membrane. The steps (a) and (b) in themethod above can be performed simultaneously or sequentially. Thus, theinvention provides methods for forming the biomimetic membrane in thepresence of one or more hydrophobins as described herein.

In some embodiments, the methods of stabilizing a biomimetic membranecontaining a membrane protein comprise the following steps: (a)providing a membrane protein; (b) adding one or more hydrophobins, asdescribed herein, to the membrane protein; and (c) adding the one ormore hydrophobins and the membrane protein to a biomimetic membrane. Insome embodiments, the membrane protein, the one or more hydrophobins andthe components that form the biomimetic membrane (e.g. lipid and/orpolymer) are added simultaneously.

In some embodiments, the methods of stabilizing a biomimetic membranecontaining a membrane protein comprise the following steps: (a)providing a biomimetic membrane containing the membrane protein; and (b)adding one or more hydrophobins, as described herein, to the biomimeticmembrane.

In some embodiments, the biomimetic membrane is a vesicle.

In some embodiments, the invention provides methods of stabilizing abiomimetic membrane containing one or more aquaporin water channels. Insome embodiments, the methods comprise adding the one or more aquaporinwater channels and one or more hydrophobins to a lipid membrane filmhydrations step during a lipid vesicle formations using any suitablemethod known in the art, including the ones described herein. In someembodiments, the methods comprise the steps of: (a) formingaquaporin-lipid vesicles, and (b) adding one or more hydrophobins to thelipid vesicles. In some embodiments, the methods comprise (a) formingaquaporin-lipid vesicles, (b) depositing the vesicles in an air/waterinterface and, (c) adding one or more hydrophobins. In some embodiments,the methods comprise forming aquaporin/hydrophobin lipid vesicles.

In some embodiments, the methods comprise adding the one or moreaquaporin water channels and one or more hydrophobins to a lipid vesicleformation step using any suitable method known in the art, including theones described herein.

In some embodiments, the methods comprise adding the one or moreaquaporin water channels and one or more hydrophobins to a polymervesicle formation step using any suitable method known in the art,including the ones described herein. In some embodiments, the methodscomprise the steps of: (a) forming aquaporin-polymer vesicles, and (b)adding one or more hydrophobins to the polymer vesicles. In someembodiments, the methods comprise (a) forming aquaporin-polymervesicles, (b) depositing the vesicles in an air/water interface and, (c)adding one or more hydrophobins. In some embodiments, the methodscomprise forming aquaporin/hydrophobin polymer vesicles.

In some embodiments, the one or more hydrophobins and the one or moreaquaporin water channels are incorporated into the lipid/polymervesicles during formation of the vesicle, by including it in thelipid/polymer solution. The one or more hydrophobins and the one or moreaquaporin water channels can also or alternatively be incorporated intothe vesicle after the vesicle has been formed. In some embodiments, theone or more hydrophobins and the one or more aquaporin water channelsare incorporated into the lipid/polymer vesicles before or after themembrane is polymerized and/or crosslinked.

In some embodiments, the one or more hydrophobins is added to thebiomimetic membrane, the membrane protein and/or a biomimetic membranecontaining the membrane protein at a concentration that is based on thecritical aggregation concentration (CAC) of the one or morehydrophobins. The term “critical aggregation concentration” or “CAC” isthe concentration above which the hydrophobins or other surfactantsaggregate or form regular shaped structures, such as micelles, nanotubesor nanovessicles. The CAC of surfactants can be determinedexperimentally using known dynamic light scattering methods. A minimalamount of sample can be used in this method. Each CAC determinationtakes a few hours, therefore it is possible to determine the CAC for alarge number of peptide detergents in a few weeks. It is known that thelower the CAC, the more hydrophobic the detergents and the stronger theaggregation in water.

In some embodiments, the one or more hydrophobins are added to thebiomimetic membrane, the membrane protein and/or a biomimetic membranecontaining the membrane protein at a concentration from about 1 timesthe CAC of the one or more hydrophobins (1×CAC) to a concentration thatis about 30 times the CAC of the one or more hydrophobins (30×CAC). Insome embodiments, the one or more hydrophobins are added to thebiomimetic membrane, the membrane protein and/or a biomimetic membranecontaining the membrane protein at a concentration which is at least 1.5times the CAC of the one or more hydrophobins (1.5×CAC). In someembodiments, the one or more hydrophobins are added at a concentrationthat is at least 2 times the CAC of the one or more hydrophobins(2×CAC). In some embodiments, the one or more hydrophobins are added ata concentration that is at least 5 times the CAC of the one or morehydrophobins (S×CAC). In some embodiments, the one or more hydrophobinsare added at a concentration that is at least 10 times the CAC of theone or more hydrophobins (10×CAC). In some embodiments, the one or morehydrophobins are added at a concentration that is at least 12 times theCAC of the one or more hydrophobins (12×CAC). In some embodiments, theone or more hydrophobins are added at a concentration that is at least15 times the CAC of the one or more hydrophobins (15×CAC). In someembodiments, the one or more hydrophobins are added at a concentrationthat is at least 20 times the CAC of the one or more hydrophobins(20×CAC).

In some embodiments, the one or more hydrophobins are added to thebiomimetic membrane, the membrane protein and/or a biomimetic membranecontaining the membrane protein on or in a dry surface. In someembodiments, the one or more hydrophobins are added to the biomimeticmembrane, the membrane protein and/or a biomimetic membrane containingthe membrane protein in an aqueous medium. In some embodiments, theaqueous medium is an aqueous solution. In a further embodiment, the oneor more hydrophobins are added to the biomimetic membrane, the membraneprotein and/or a biomimetic membrane containing the membrane protein inan ionic solution. In some embodiments, the method comprises theaddition of a single type of hydrophobin. In some embodiments, themethod comprises the addition of at least two different hydrophobins.

In some embodiments, a surfactant peptide and/or a non-peptidesurfactant can be added to the biomimetic membrane, the membrane proteinand/or a biomimetic membrane containing the membrane protein. Asurfactant is a compound that is amphiphilic or that contains bothhydrophobic groups (their “tails”) and hydrophilic groups (their“heads”). Surfactants are soluble in both organic solvents and water.There are generally two types of surfactants, ionic and non-ionicsurfactants. Ionic surfactants are surfactants that have a net charge attheir heads. Non-ionic surfactants are surfactants that have no netcharge at their heads. Examples of non-peptide surfactants include, butare not limited to polyoxyalkylene sorbitan fatty acid esters, sorbitanfatty acid esters, alkylene glycol fatty acid esters, polyoxyalkylenefatty acid esters, fatty acid esters, polyoxyalkylene fatty acid ethers,C₁₆C₂₄ fatty acids, fatty acid mono-, di- or poly-glycerides,polyoxyalkylene alkyl phenols, alkyl phenyl ethers, polyoxyethylenepolyoxypropylene block copolymers, fatty amine oxides, fatty acidalkanolamides, alkyl cellulose, carboxyalkyl cellulose andpolyoxyalkylene castor oil derivatives. Ionic surfactants include, butare not limited to, alkyl sulfates, olefin sulfates, ether sulfates,monoglyceride sulfates, alkyl sulfonates, aryl sulfonates, olefinsulfonates, alkyl sulfosuccinates, aryl sulfosuccinates, includingsodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodiumsulfonate), benzalkonium salts, polyoxyalkylene alkylamines,alkylamines, alkanolamine fatty acid esters, quaternary ammonium fattyacid esters, dialkyl ammonium salts, alkyl pyridinium salts includingstearylamine and triethanolamine oleate, benzethonium chloride.Non-limiting examples of non-peptide surfactant are lauryldimethyamineoxide (LDAO), n-dodecyldimethyamine N-oxide (NDAO), OctyldimethyamineN-oxide (ODAO), undecyldimethyamine N-oxide (UDAO),Octyl-.beta.-D-glucose (beta-OG), Decyl-beta-D-glucose (beta-DG),Nonyl-beta-D-glucose (beta-NG), Dodecyl-beta-D-maltoside (DDM),Octyanoylsucrose (OS), Octyl-beta-D-galactoside (beta-OGal) and Dodecylphosphocholine (DPC). In some embodiments, the non-peptide surfactantused in the method of the invention is a non-ionic surfactant. In afurther embodiment, the non-ionic surfactant is selected from the groupconsisting of n-dodecyl-B-D-maltoside and octyl-D-glucoside. In someembodiments, the non-peptide surfactant is added in an amount betweenabout 2 and about 200 times the CAC of the non-peptide surfactant.

In some embodiments, addition of one or more hydrophobins provides atleast about a 1.2-fold increase in stability of a biomimetic membrane, amembrane protein and/or a biomimetic membrane containing the membraneprotein compared to the stability in the absence of the one or morehydrophobins. In another embodiment, addition of one or morehydrophobins provides at least about 1.5, at least about 1.7, at leastabout 2.0, at least about 2.2, at least about 2.3 or at least about2.5-fold increase in stability a biomimetic membrane, a membrane proteinand/or a biomimetic membrane containing the membrane protein compared tothe stability in the absence of the one or more hydrophobins.

In some embodiments, addition of one or more hydrophobins provides adecrease in the equilibrium surface tension at a biomimetic membrane(e.g. a biomimetic membrane containing one or more membrane proteins)below 50 mN/m. In some embodiments, such a reduction in the equilibriumsurface tension at a biomimetic membrane may be achieved using one ormore hydrophobins at a concentration of between 0.01-100 mM, 0.01-50 mMor 0.01-20 mM. In some embodiments, the hydrophobin is present in aconcentration of 0.1 μM-50 mM. In some embodiments, the hydrophobin ispresent in a concentration of 0.1-20% by weight of the total weight ofthe composition. In some embodiments such a reduction in the equilibriumsurface tension at a biomimetic membrane may be achieved at atemperature ranging from 0-90° C.

In some embodiments, the one or more hydrophobins may cause the surfaceshear elasticity at a biomimetic membrane (e.g. a biomimetic membranecontaining one or more membrane proteins) to increase to 30-35 mN/m,40-50 mN/m, or higher. In some embodiments, the one or more hydrophobinsmay cause the surface shear elasticity at a biomimetic membrane (e.g. abiomimetic membrane containing one or more membrane proteins) toincrease to 0.3-0.6 N/m, or higher. In some embodiments, such surfaceshear elasticity at a biomimetic membrane may be achieved using one ormore hydrophobins at a concentration of between 0.01-100 mM, 0.01-50 mMor 0.01-20 mM. In some embodiments, the hydrophobin is present in aconcentration of 0.1 μM-50 mM. In some embodiments, the hydrophobin ispresent in a concentration of 0.1-20% by weight of the total weight ofthe composition. In some embodiments, such surface shear elasticity at abiomimetic membrane may be achieved at a temperature ranging from 0-90°C.

Kits

The present invention also encompasses kits for stabilizing membraneproteins and/or biomimetic membranes. The kit comprises a compositioncomprising a membrane protein and a composition comprising one or morehydrophobins as described herein. Kits according to the invention mayfurther comprise a composition comprising a peptide surfactant and/ornon-peptide surfactant.

In some embodiments, the kit comprises one or more components for theformation of a biomimetic membrane. In some embodiments, the kitcomprises one or more biomimetic membranes.

The kit may further comprise one or more other components for aparticular application (e.g. water filtering device).

Host Cell

The term “host cell”—in relation to the present invention includes anycell that comprises either a nucleotide sequence or an expression vectoras described herein and which is used in the recombinant production of aprotein having the specific properties as defined herein.

Thus, a further embodiment of the present invention provides host cellstransformed or transfected with a nucleotide sequence that expresses theprotein(s) of the present invention. The cells will be chosen to becompatible with the said vector and may for example be prokaryotic (forexample bacterial), fungal, yeast or plant cells. Preferably, the hostcells are not human cells.

Examples of suitable bacterial host organisms are gram positive or gramnegative bacterial species.

Depending on the nature of the nucleotide sequence encoding the proteinof the present invention, and/or the desirability for further processingof the expressed protein, eukaryotic hosts such as yeasts or other fungimay be preferred. However, some proteins are either poorly secreted fromthe yeast cell, or in some cases are not processed properly (e.g.,hyper-glycosylation in yeast).

The use of suitable host cells—such as yeast, fungal and plant hostcells—may provide for post-translational modifications (e.g.,myristoylation, glycosylation, truncation, lipidation and tyrosine,serine or threonine phosphorylation, or N-terminal acetylation as may beneeded to confer optimal biological activity on recombinant expressionproducts of the present invention.

The host cell may be a protease deficient or protease minus strain.

The genotype of the host cell may be modified to improve expression.

Examples of host cell modifications include protease deficiency,supplementation of rare tRNAs, and modification of the reductivepotential in the cytoplasm to enhance disulphide bond formation.

For example, the host cell E. coli may overexpress rare tRNAs to improveexpression of heterologous proteins as exemplified/described in Kane(Curr Opin Biotechnol (1995), 6, 494-500 “Effects of rare codon clusterson high-level expression of heterologous proteins in E. coli”). The hostcell may be deficient in a number of reducing enzymes thus favoringformation of stable disulphide bonds as exemplified/described inBessette (Proc Natl Acad Sci USA (1999), 96, 13703-13708 “Efficientfolding of proteins with multiple disulphide bonds in the Escherichiacoli cytoplasm”).

Isolated

In one aspect, the polypeptide(s) for use in the present invention maybe in an isolated form. The terms “polypeptide”, “protein”, “peptide”and “amino acid sequence” are used herein interchangeably.

The term “isolated” means that the sequence or protein is at leastsubstantially free from at least one other component with which thesequence or protein is naturally associated in nature and as found innature.

Purified

In one aspect, the polypeptide(s) for use in the present invention maybe used in a purified form.

The term “purified” means that the sequence is in a relatively purestate—e.g., at least about 51% pure, or at least about 75%, or at leastabout 80%, or at least about 90% pure, or at least about 95% pure or atleast about 98% pure.

Cloning a Nucleotide Sequence Encoding a Polypeptide According to thePresent Invention

A nucleotide sequence encoding either a polypeptide which has thespecific properties as defined herein or a polypeptide which is suitablefor modification may be isolated from any cell or organism producingsaid polypeptide. Various methods are well known within the art for theisolation of nucleotide sequences.

For example, a genomic DNA and/or cDNA library may be constructed usingchromosomal DNA or messenger RNA from the organism producing thepolypeptide. If the amino acid sequence of the polypeptide is known,labeled oligonucleotide probes may be synthesized and used to identifypolypeptide-encoding clones from the genomic library prepared from theorganism. Alternatively, a labeled oligonucleotide probe containingsequences homologous to another known polypeptide gene could be used toidentify polypeptide-encoding clones. In the latter case, hybridizationand washing conditions of lower stringency are used.

Alternatively, polypeptide-encoding clones could be identified byinserting fragments of genomic DNA into an expression vector, such as aplasmid, transforming enzyme-negative bacteria with the resultinggenomic DNA library, and then plating the transformed bacteria onto agarcontaining an enzyme inhibited by the polypeptide, thereby allowingclones expressing the polypeptide to be identified.

In a yet further alternative, the nucleotide sequence encoding thepolypeptide may be prepared synthetically by established standardmethods, e.g., the phosphoroamidite method described by Beucage S. L. etal. (1981) Tetrahedron Letters 22, 1859-1869, or the method described byMatthes et al. (1984) EMBO J. 3, 801-805. In the phosphoroamiditemethod, oligonucleotides are synthesized, e.g., in an automatic DNAsynthesizer, purified, annealed, ligated and cloned in appropriatevectors.

The nucleotide sequence may be of mixed genomic and synthetic origin,mixed synthetic and cDNA origin, or mixed genomic and cDNA origin,prepared by ligating fragments of synthetic, genomic or cDNA origin (asappropriate) in accordance with standard techniques. Each ligatedfragment corresponds to various parts of the entire nucleotide sequence.The DNA sequence may also be prepared by polymerase chain reaction (PCR)using specific primers, for instance as described in U.S. Pat. No.4,683,202 or in Saiki R K et al. (Science (1988) 239, 487-491).

Nucleotide Sequences

The present invention also encompasses nucleotide sequences encodingpolypeptides having the specific properties as defined herein. The term“nucleotide sequence” as used herein refers to an oligonucleotidesequence or polynucleotide sequence, and variant, homologues, fragmentsand derivatives thereof (such as portions thereof). The nucleotidesequence may be of genomic or synthetic or recombinant origin, which maybe double-stranded or single-stranded whether representing the sense orantisense strand.

The term “nucleotide sequence” in relation to the present inventionincludes genomic DNA, cDNA, synthetic DNA, and RNA. Preferably it meansDNA, more preferably cDNA for the coding sequence.

In some embodiments, the nucleotide sequence per se encoding apolypeptide having the specific properties as defined herein does notcover the native nucleotide sequence in its natural environment when itis linked to its naturally associated sequence(s) that is/are also inits/their natural environment. For ease of reference, we shall call thisembodiment the “non-native nucleotide sequence”. In this regard, theterm “native nucleotide sequence” means an entire nucleotide sequencethat is in its native environment and when operatively linked to anentire promoter with which it is naturally associated, which promoter isalso in its native environment.

However, the amino acid sequence encompassed by scope the presentinvention can be isolated and/or purified post expression of anucleotide sequence in its native organism. In some embodiments,however, the amino acid sequence encompassed by scope of the presentinvention may be expressed by a nucleotide sequence in its nativeorganism but wherein the nucleotide sequence is not under the control ofthe promoter with which it is naturally associated within that organism.

In some embodiments, the polypeptide is not a native polypeptide. Inthis regard, the term “native polypeptide” means an entire polypeptidethat is in its native environment and when it has been expressed by itsnative nucleotide sequence.

Typically, the nucleotide sequence encoding polypeptides having thespecific properties as defined herein is prepared using recombinant DNAtechniques (i.e., recombinant DNA). However, in an alternativeembodiment of the invention, the nucleotide sequence could besynthesized, in whole or in part, using chemical methods well known inthe art (see Caruthers M H et al. (1980) Nuc Acids Res Symp Ser 215-23and Horn T et al. (1980) Nuc Acids Res Symp Ser 225-232).

Molecular Evolution

Once a polypeptide-encoding nucleotide sequence has been isolated, or aputative polypeptide-encoding nucleotide sequence has been identified,it may be desirable to modify the selected nucleotide sequence, forexample it may be desirable to mutate the sequence in order to prepare apolypeptide in accordance with the present invention.

Mutations may be introduced using synthetic oligonucleotides. Theseoligonucleotides contain nucleotide sequences flanking the desiredmutation sites.

A suitable method is disclosed in Morinaga et al. (Biotechnology (1984)2, 646-649). Another method of introducing mutations intopolypeptide-encoding nucleotide sequences is described in Nelson andLong (Analytical Biochemistry (1989), 180, 147-151).

Instead of site directed mutagenesis, such as described above, one canintroduce mutations randomly for instance using a commercial kit such asthe GeneMorph PCR mutagenesis kit from Stratagene, or the Diversify PCRrandom mutagenesis kit from Clontech. EP 0 583 265 refers to methods ofoptimizing PCR based mutagenesis, which can also be combined with theuse of mutagenic DNA analogues such as those described in EP 0 866 796.Error prone PCR technologies are suitable for the production of variantsof polypeptides with preferred characteristics.

A third method to obtain novel sequences is to fragment non-identicalnucleotide sequences, either by using any number of restriction enzymesor an enzyme such as Dnase I, and reassembling full nucleotide sequencescoding for functional proteins. Alternatively one can use one ormultiple non-identical nucleotide sequences and introduce mutationsduring the reassembly of the full nucleotide sequence. DNA shuffling andfamily shuffling technologies are suitable for the production ofvariants of polypeptides with preferred characteristics. Suitablemethods for performing ‘shuffling’ can be found in EP 0 752 008, EP 1138 763, EP 1 103 606. Shuffling can also be combined with other formsof DNA mutagenesis as described in U.S. Pat. No. 6,180,406 and WO01/34835.

Thus, it is possible to produce numerous site directed or randommutations into a nucleotide sequence, either in vivo or in vitro, and tosubsequently screen for improved functionality of the encodedpolypeptide by various means. Using in silico and exo-mediatedrecombination methods (see, e.g., WO 00/58517, U.S. Pat. No. 6,344,328,U.S. Pat. No. 6,361,974), for example, molecular evolution can beperformed where the variant produced retains very low homology to knownproteins. Such variants thereby obtained may have significant structuralanalogy to known proteins, but have very low amino acid sequencehomology.

As a non-limiting example, in addition, mutations or natural variants ofa polynucleotide sequence can be recombined with either the wild type orother mutations or natural variants to produce new variants. Such newvariants can also be screened for improved functionality of the encodedpolypeptide.

The application of the above-mentioned and similar molecular evolutionmethods allows the identification and selection of variants of thepolypeptides of the present invention which have preferredcharacteristics without any prior knowledge of protein structure orfunction, and allows the production of non-predictable but beneficialmutations or variants. There are numerous examples of the application ofmolecular evolution in the art for the optimization or alteration ofprotein activity, such examples include, but are not limited to one ormore of the following: optimized expression and/or activity in a hostcell or in vitro, increased or decreased enzymatic activity, alteredsubstrate and/or product specificity, increased or decreased structuralstability, altered activity/specificity in preferred environmentalconditions, e.g., temperature, pH, substrate.

As will be apparent to a person skilled in the art, using molecularevolution tools a polypeptide may be altered to improve thefunctionality of the polypeptide

Alternatively, the variant polypeptide may have increasedthermostability.

Amino Acid Sequences

The present invention also encompasses the use of amino acid sequencesencoded by a nucleotide sequence which encodes a polypeptide for use inany one of the methods and/or uses of the present invention.

As used herein, the term “amino acid sequence” is synonymous with theterm “polypeptide” and/or the term “protein”. In some instances, theterm “amino acid sequence” is synonymous with the term “peptide”.

The amino acid sequence may be prepared/isolated from a suitable source,or it may be made synthetically or it may be prepared by use ofrecombinant DNA techniques.

Suitably, the amino acid sequences may be obtained from the isolatedpolypeptides taught herein by standard techniques.

One suitable method for determining amino acid sequences from isolatedpolypeptides is as follows:

Purified polypeptide may be freeze-dried and 100 μg of the freeze-driedmaterial may be dissolved in 50 μl of a mixture of 8 M urea and 0.4 Mammonium hydrogen carbonate, pH 8.4. The dissolved protein may bedenatured and reduced for 15 minutes at 50° C. following overlay withnitrogen and addition of 5 μl of 45 mM dithiothreitol. After cooling toroom temperature, 5 μl of 100 mM iodoacetamide may be added for thecysteine residues to be derivatized for 15 minutes at room temperaturein the dark under nitrogen. 135 μl of water and 5 μg of endoproteinaseLys-C in 5 μl of water may be added to the above reaction mixture andthe digestion may be carried out at 37° C. under nitrogen for 24 hours.The resulting peptides may be separated by reverse phase HPLC on a VYDACC18 column (0.46×15 cm; 10 μm; The Separation Group, California, USA)using solvent A: 0.1% TFA in water and solvent B: 0.1% TFA inacetonitrile. Selected peptides may be re-chromatographed on a DevelosilC18 column using the same solvent system, prior to N-terminalsequencing. Sequencing may be done using an Applied Biosystems 476Asequencer using pulsed liquid fast cycles according to themanufacturer's instructions (Life Technologies, California, USA).

Sequence Identity or Sequence Homology

Here, the term “homologue” means an entity having a certain homologywith the subject amino acid sequences and the subject nucleotidesequences. Here, the term “homology” can be equated with “identity”.

In some embodiments, the homologous amino acid sequence and/ornucleotide sequence provide and/or encode a polypeptide which retainsthe desire characteristic(s) and/or enhances the desirecharacteristic(s) of the polypeptide.

In the present context, a homologous sequence is taken to include anamino acid sequence which may be at least 50%, 55%, 60%, 70%, 71%, 72%,73%, 74%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical, preferably at least 95%, 96%, 97%, 98%, or 99% identicalto the subject sequence. Typically, the homologues will comprise thesame active sites etc. as the subject amino acid sequence. Althoughhomology can also be considered in terms of similarity (i.e., amino acidresidues having similar chemical properties/functions), in the contextof the present invention it is preferred to express homology in terms ofsequence identity.

In the present context, a homologous sequence is taken to include anucleotide sequence which may be at least 75, 85 or 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical, preferably at least 95%, 96%,97%, 98%, or 99% identical to a nucleotide sequence encoding apolypeptide of the present invention (the subject sequence). In someembodiments, the homologues will comprise the same sequences that codefor the active sites as the subject sequence. Although homology can alsobe considered in terms of similarity (e.g., amino acid residues havingsimilar chemical properties/functions), in the context of the presentinvention it is preferred to express homology in terms of sequenceidentity.

Homology comparisons can be conducted by eye, or more usually, with theaid of readily available sequence comparison programs. Thesecommercially available computer programs can calculate % homologybetween two or more sequences.

% homology may be calculated over contiguous sequences, i.e., onesequence is aligned with the other sequence and each amino acid in onesequence is directly compared with the corresponding amino acid in theother sequence, one residue at a time. This is called an “ungapped”alignment. Typically, such ungapped alignments are performed only over arelatively short number of residues.

Although this is a very simple and consistent method, it fails to takeinto consideration that, for example, in an otherwise identical pair ofsequences, one insertion or deletion will cause the following amino acidresidues to be put out of alignment, thus potentially resulting in alarge reduction in % homology when a global alignment is performed.Consequently, most sequence comparison methods are designed to produceoptimal alignments that take into consideration possible insertions anddeletions without penalizing unduly the overall homology score. This isachieved by inserting “gaps” in the sequence alignment to try tomaximize local homology.

However, these more complex methods assign “gap penalties” to each gapthat occurs in the alignment so that, for the same number of identicalamino acids, a sequence alignment with as few gaps aspossible—reflecting higher relatedness between the two comparedsequences—will achieve a higher score than one with many gaps. “Affinegap costs” are typically used that charge a relatively high cost for theexistence of a gap and a smaller penalty for each subsequent residue inthe gap. This is the most commonly used gap scoring system. High gappenalties will of course produce optimized alignments with fewer gaps.Most alignment programs allow the gap penalties to be modified. However,it is preferred to use the default values when using such software forsequence comparisons.

Calculation of maximum % homology therefore firstly requires theproduction of an optimal alignment, taking into consideration gappenalties. A suitable computer program for carrying out such analignment is the Vector NTI (Invitrogen Corp.). Examples of othersoftware that can perform sequence comparisons include, but are notlimited to, the BLAST package (see Ausubel et al. 1999 Short Protocolsin Molecular Biology, 4^(th) Ed—Chapter 18), and FASTA (Altschul et al.1990 J. Mol. Biol. 403-410). Both BLAST and FASTA are available foroffline and online searching (see Ausubel et al. 1999, pages 7-58 to7-60). However, for some applications, it is preferred to use the VectorNTI program. A new tool, called BLAST 2 Sequences is also available forcomparing protein and nucleotide sequence (see FEMS Microbiol Lett 1999174: 247-50; FEMS Microbiol Lett 1999 177: 187-8).

Although the final % homology can be measured in terms of identity, thealignment process itself is typically not based on an all-or-nothingpair comparison. Instead, a scaled similarity score matrix is generallyused that assigns scores to each pairwise comparison based on chemicalsimilarity or evolutionary distance. An example of such a matrixcommonly used is the BLOSUM62 matrix—the default matrix for the BLASTsuite of programs. Vector NTI programs generally use either the publicdefault values or a custom symbol comparison table if supplied (see usermanual for further details). For some applications, it is preferred touse the default values for the Vector NTI ADVANCE™ 10 package.

Alternatively, percentage homologies may be calculated using themultiple alignment feature in Vector NTI ADVANCE™ 10 (Invitrogen Corp.),based on an algorithm, analogous to CLUSTAL (Higgins DG & Sharp PM(1988), Gene 73, 237-244).

Once the software has produced an optimal alignment, it is possible tocalculate % homology, preferably % sequence identity. The softwaretypically does this as part of the sequence comparison and generates anumerical result.

Suitably, the degree of identity with regard to a nucleotide sequence isdetermined over at least 20 contiguous nucleotides, preferably over atleast 30 contiguous nucleotides, preferably over at least 40 contiguousnucleotides, preferably over at least 50 contiguous nucleotides,preferably over at least 60 contiguous nucleotides, preferably over atleast 100 contiguous nucleotides.

Suitably, the degree of identity with regard to a nucleotide sequencemay be determined over the whole sequence.

Should Gap Penalties be used when determining sequence identity, thenpreferably the default parameters for the program are used for pairwisealignment. For example, the following parameters are the current defaultparameters for pairwise alignment for BLAST 2:

FOR BLAST2 DNA PROTEIN EXPECT THRESHOLD 10 10 WORD SIZE 11  3 SCORINGPARAMETERS Match/Mismatch Scores 2, −3 n/a Matrix n/a BLOSUM62 Gap CostsExistence: 5 Existence: 11 Extension: 2 Extension: 1

In some embodiments, the sequence identity for the nucleotide sequencesand/or amino acid sequences may be determined using BLAST2 (blastn) withthe scoring parameters set as defined above.

In some embodiments, the degree of identity is based on the number ofsequence elements which are the same. The degree of identity inaccordance with the present invention for amino acid sequences may besuitably determined by means of computer programs known in the art suchas Vector NTI ADVANCE™ 11 (Invitrogen Corp.). For pairwise alignment thescoring parameters used are preferably BLOSUM62 with Gap existencepenalty of 11 and Gap extension penalty of 1.

Suitably, the degree of identity with regard to an amino acid sequenceis determined over at least 20 contiguous amino acids, preferably overat least 30 contiguous amino acids, preferably over at least 40contiguous amino acids, preferably over at least 50 contiguous aminoacids, preferably over at least 60 contiguous amino acids, preferablyover at least 100 contiguous amino acids.

Suitably, the degree of identity with regard to an amino acid sequencemay be determined over the whole sequence.

The sequences may also have deletions, insertions or substitutions ofamino acid residues which produce a silent change and result in afunctionally equivalent substance. Deliberate amino acid substitutionsmay be made on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues as long as the secondary binding activity of the substance isretained. For example, negatively charged amino acids include asparticacid and glutamic acid; positively charged amino acids include lysineand arginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values include leucine, isoleucine, valine,glycine, alanine, asparagine, glutamine, serine, threonine,phenylalanine, and tyrosine.

Conservative substitutions may be made, for example according to theTable below. Amino acids in the same block in the second column andpreferably in the same line in the third column may be substituted foreach other:

ALIPHATIC Non-polar G A P I L V Polar - uncharged C S T M N Q Polar -charged D E K R AROMATIC H F W Y

The present invention also encompasses homologous substitution(substitution and replacement are both used herein to mean theinterchange of an existing amino acid residue, with an alternativeresidue) that may occur, e.g., like-for-like substitution such as basicfor basic, acidic for acidic, polar for polar etc. Non-homologoussubstitution may also occur, e.g., from one class of residue to anotheror alternatively involving the inclusion of unnatural amino acids suchas ornithine (hereinafter referred to as Z), diaminobutyric acidornithine (hereinafter referred to as B), norleucine ornithine(hereinafter referred to as O), pyridylalanine, thienylalanine,naphthylalanine and phenylglycine.

Replacements may also be made by non-natural amino acids.

Variant amino acid sequences may include suitable spacer groups that maybe inserted between any two amino acid residues of the sequenceincluding alkyl groups such as methyl, ethyl or propyl groups inaddition to amino acid spacers such as glycine or β-alanine residues. Afurther form of variation, involves the presence of one or more aminoacid residues in peptoid form, will be well understood by those skilledin the art. For the avoidance of doubt, “the peptoid form” is used torefer to variant amino acid residues wherein the α-carbon substituentgroup is on the residue's nitrogen atom rather than the α-carbon.Processes for preparing peptides in the peptoid form are known in theart, for example Simon R J et al., PNAS (1992) 89, 9367-9371 and HorwellD C, Trends Biotechnol. (1995) 13, 132-134.

Nucleotide sequences for use in the present invention or encoding apolypeptide having the specific properties defined herein may includewithin them synthetic or modified nucleotides. A number of differenttypes of modification to oligonucleotides are known in the art. Theseinclude methylphosphonate and phosphorothioate backbones and/or theaddition of acridine or polylysine chains at the 3′ and/or 5′ ends ofthe molecule. For the purposes of the present invention, it is to beunderstood that the nucleotide sequences described herein may bemodified by any method available in the art. Such modifications may becarried out in order to enhance the in vivo activity or life span ofnucleotide sequences.

The present invention also encompasses the use of nucleotide sequencesthat are complementary to the sequences discussed herein, or anyderivative, fragment or derivative thereof. If the sequence iscomplementary to a fragment thereof then that sequence can be used as aprobe to identify similar coding sequences in other organisms etc.

Polynucleotides which are not 100% homologous to the sequences of thepresent invention but fall within the scope of the invention can beobtained in a number of ways. Other variants of the sequences describedherein may be obtained for example by probing DNA libraries made from arange of individuals, for example individuals from differentpopulations. In addition, other viral/bacterial, or cellular homologuesparticularly cellular homologues found in mammalian cells (e.g., rat,mouse, bovine and primate cells), may be obtained and such homologuesand fragments thereof in general will be capable of selectivelyhybridizing to the sequences shown in the sequence listing herein. Suchsequences may be obtained by probing cDNA libraries made from or genomicDNA libraries from other animal species, and probing such libraries withprobes comprising all or part of any one of the sequences in theattached sequence listings under conditions of medium to highstringency. Similar considerations apply to obtaining species homologuesand allelic variants of the polypeptide or nucleotide sequences of theinvention.

Variants and strain/species homologues may also be obtained usingdegenerate PCR which will use primers designed to target sequenceswithin the variants and homologues encoding conserved amino acidsequences within the sequences of the present invention. Conservedsequences can be predicted, for example, by aligning the amino acidsequences from several variants/homologues. Sequence alignments can beperformed using computer software known in the art. For example the GCGWisconsin PileUp program is widely used.

The primers used in degenerate PCR will contain one or more degeneratepositions and will be used at stringency conditions lower than thoseused for cloning sequences with single sequence primers against knownsequences.

Alternatively, such polynucleotides may be obtained by site directedmutagenesis of characterized sequences. This may be useful where forexample silent codon sequence changes are required to optimize codonpreferences for a particular host cell in which the polynucleotidesequences are being expressed. Other sequence changes may be desired inorder to introduce restriction polypeptide recognition sites, or toalter the property or function of the polypeptides encoded by thepolynucleotides.

Polynucleotides (nucleotide sequences) of the invention may be used toproduce a primer, e.g., a PCR primer, a primer for an alternativeamplification reaction, a probe e.g., labeled with a revealing label byconventional means using radioactive or non-radioactive labels, or thepolynucleotides may be cloned into vectors. Such primers, probes andother fragments will be at least 15, preferably at least 20, for exampleat least 25, 30 or 40 nucleotides in length, and are also encompassed bythe term polynucleotides of the invention as used herein.

Polynucleotides such as DNA polynucleotides and probes according to theinvention may be produced recombinantly, synthetically, or by anysuitable means available to those of skill in the art. They may also becloned by standard techniques.

In some embodiments, primers will be produced by synthetic means,involving a stepwise manufacture of the desired nucleic acid sequenceone nucleotide at a time. Techniques for accomplishing these automatedtechniques are readily available in the art.

In some embodiments, longer polynucleotides will generally be producedusing recombinant means, for example using a PCR (polymerase chainreaction) cloning techniques. This will involve making a pair of primers(e.g., of about 15 to 30 nucleotides) flanking a region of the targetsequence which it is desired to clone, bringing the primers into contactwith mRNA or cDNA obtained from an animal or human cell, performing apolymerase chain reaction under conditions which bring aboutamplification of the desired region, isolating the amplified fragment(e.g., by purifying the reaction mixture on an agarose gel) andrecovering the amplified DNA. The primers may be designed to containsuitable restriction enzyme recognition sites so that the amplified DNAcan be cloned into a suitable cloning vector.

Hybridization

The present invention also encompasses the use of sequences that arecomplementary to the sequences of the present invention or sequencesthat are capable of hybridizing either to the sequences of the presentinvention or to sequences that are complementary thereto.

The term “hybridization” as used herein shall include “the process bywhich a strand of nucleic acid joins with a complementary strand throughbase pairing” as well as the process of amplification as carried out inpolymerase chain reaction (PCR) technologies.

The present invention also encompasses the use of nucleotide sequencesthat are capable of hybridizing to the sequences that are complementaryto the subject sequences discussed herein, or any derivative, fragmentor derivative thereof.

The present invention also encompasses sequences that are complementaryto sequences that are capable of hybridizing to the nucleotide sequencesdiscussed herein.

Hybridization conditions are based on the melting temperature (Tm) ofthe nucleotide binding complex, as taught in Berger and Kimmel (1987,Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol. 152,Academic Press, San Diego Calif.), and confer a defined “stringency” asexplained below.

Maximum stringency typically occurs at about Tm-5° C. (5° C. below theTm of the probe); high stringency at about 5° C. to 10° C. below Tm;intermediate stringency at about 10° C. to 20° C. below Tm; and lowstringency at about 20° C. to 25° C. below Tm. As will be understood bythose of skill in the art, a maximum stringency hybridization can beused to identify or detect identical nucleotide sequences while anintermediate (or low) stringency hybridization can be used to identifyor detect similar or related polynucleotide sequences.

In some embodiments, the present invention encompasses the use ofsequences that are complementary to sequences that are capable ofhybridizing under high stringency conditions or intermediate stringencyconditions to nucleotide sequences encoding polypeptides having thespecific properties as defined herein.

In some embodiments, the present invention encompasses the use ofsequences that are complementary to sequences that are capable ofhybridizing under high stringency conditions (e.g., 65° C. and 0.1×SSC{1×SSC=0.15 M NaCl, 0.015 M Na-citrate pH 7.0}) to nucleotide sequencesencoding polypeptides having the specific properties as defined herein.

The present invention also relates to the use of nucleotide sequencesthat can hybridize to the nucleotide sequences discussed herein(including complementary sequences of those discussed herein).

The present invention also relates to the use of nucleotide sequencesthat are complementary to sequences that can hybridize to the nucleotidesequences discussed herein (including complementary sequences of thosediscussed herein).

Also included within the scope of the present invention is the use ofpolynucleotide sequences that are capable of hybridizing to thenucleotide sequences discussed herein under conditions of intermediateto maximal stringency.

In some embodiments, the present invention covers the use of nucleotidesequences that can hybridize to the nucleotide sequences discussedherein, or the complement thereof, under stringent conditions (e.g., 50°C. and 0.2×SSC).

In some embodiments, the present invention covers the use of nucleotidesequences that can hybridize to the nucleotide sequences discussedherein, or the complement thereof, under high stringency conditions(e.g., 65° C. and 0.1×SSC).

Biologically Active

Preferably, the variant sequences etc. are at least as biologicallyactive as the sequences presented herein.

As used herein “biologically active” refers to a sequence having asimilar structural function (but not necessarily to the same degree),and/or similar regulatory function (but not necessarily to the samedegree), and/or similar biochemical function (but not necessarily to thesame degree) of the naturally occurring sequence.

Recombinant

In some embodiments, the sequence for use in the present invention is arecombinant sequence—i.e., a sequence that has been prepared usingrecombinant DNA techniques.

These recombinant DNA techniques are within the capabilities of a personof ordinary skill in the art. Such techniques are explained in theliterature, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis,1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3,Cold Spring Harbor Laboratory Press.

Synthetic

In s the sequence for use in the present invention is a syntheticsequence—i.e., a sequence that has been prepared by in vitro chemical orenzymatic synthesis. It includes, but is not limited to, sequences madewith optimal codon usage for host organisms—such as the methylotrophicyeasts Pichia and Hansenula.

Expression of Polypeptides

A nucleotide sequence for use in the present invention or for encoding apolypeptide having the specific properties as defined herein can beincorporated into a recombinant replicable vector. The vector may beused to replicate and express the nucleotide sequence, in polypeptideform, in and/or from a compatible host cell.

Expression may be controlled using control sequences which includepromoters/enhancers and other expression regulation signals. Prokaryoticpromoters and promoters functional in eukaryotic cells may be used.Tissue specific or stimuli specific promoters may be used. Chimericpromoters may also be used comprising sequence elements from two or moredifferent promoters described above.

The polypeptide produced by a host recombinant cell by expression of thenucleotide sequence may be secreted or may be contained intracellularlydepending on the sequence and/or the vector used. The coding sequencescan be designed with signal sequences which direct secretion of thesubstance coding sequences through a particular prokaryotic oreukaryotic cell membrane.

Expression Vector

The term “expression vector” means a construct capable of in vivo or invitro expression.

In some embodiments, the expression vector is incorporated into thegenome of a suitable host organism. The term “incorporated” preferablycovers stable incorporation into the genome.

The nucleotide sequence encoding a polypeptide for use in the presentinvention may be present in a vector in which the nucleotide sequence isoperably linked to regulatory sequences capable of providing for theexpression of the nucleotide sequence by a suitable host organism.

The vectors for use in the present invention may be transformed into asuitable host cell as described below to provide for expression of apolypeptide of the present invention.

The choice of vector e.g., a plasmid, cosmid, or phage vector will oftendepend on the host cell into which it is to be introduced.

The vectors for use in the present invention may contain one or moreselectable marker genes such as a gene which confers antibioticresistance e.g., ampicillin, kanamycin, chloramphenicol or tetracyclineresistance. Alternatively, the selection may be accomplished byco-transformation (as described in WO 91/17243).

Vectors may be used in vitro, for example for the production of RNA orused to transfect, transform, transduce or infect a host cell.

The vector may further comprise a nucleotide sequence enabling thevector to replicate in the host cell in question. Examples of suchsequences are the origins of replication of plasmids pUC19, pACYC177,pUB110, pE194, pAMB1 and pIJ702.

Regulatory Sequences

In some embodiments, the nucleotide sequence for use in the presentinvention is operably linked to a regulatory sequence which is capableof providing for the expression of the nucleotide sequence, such as bythe chosen host cell. By way of example, the present invention covers avector comprising the nucleotide sequence of the present inventionoperably linked to such a regulatory sequence, i.e., the vector is anexpression vector.

The term “operably linked” refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner A regulatory sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under condition compatible with the controlsequences.

The term “regulatory sequences” includes promoters and enhancers andother expression regulation signals.

The term “promoter” is used in the normal sense of the art, e.g. an RNApolymerase binding site.

Enhanced expression of the nucleotide sequence encoding the enzyme ofthe present invention may also be achieved by the selection ofheterologous regulatory regions, e.g., promoter, secretion leader andterminator regions.

Preferably, the nucleotide sequence according to the present inventionis operably linked to at least a promoter.

Examples of suitable promoters for directing the transcription of thenucleotide sequence in a bacterial, fungal or yeast host are well knownin the art.

Constructs

The term “construct”—which is synonymous with terms such as “conjugate”,“cassette” and “hybrid”—includes a nucleotide sequence encoding apolypeptide having the specific properties as defined herein for useaccording to the present invention directly or indirectly attached to apromoter. An example of an indirect attachment is the provision of asuitable spacer group such as an intron sequence, such as the Sh1-intronor the ADH intron, intermediate the promoter and the nucleotide sequenceof the present invention. The same is true for the term “fused” inrelation to the present invention which includes direct or indirectattachment. In some cases, the terms do not cover the naturalcombination of the nucleotide sequence coding for the protein ordinarilyassociated with the wild type gene promoter and when they are both intheir natural environment.

The construct may even contain or express a marker which allows for theselection of the genetic construct.

For some applications, preferably the construct comprises at least anucleotide sequence of the present invention or a nucleotide sequenceencoding a polypeptide having the specific properties as defined hereinoperably linked to a promoter.

Organism

The term “organism” in relation to the present invention includes anyorganism that could comprise a nucleotide sequence according to thepresent invention or a nucleotide sequence encoding for a polypeptidehaving the specific properties as defined herein and/or productsobtained therefrom.

The term “transgenic organism” in relation to the present inventionincludes any organism that comprises a nucleotide sequence coding for apolypeptide having the specific properties as defined herein and/or theproducts obtained therefrom, and/or wherein a promoter can allowexpression of the nucleotide sequence coding for a polypeptide havingthe specific properties as defined herein within the organism.Preferably the nucleotide sequence is incorporated in the genome of theorganism.

Suitable organisms include a prokaryote, fungus yeast or a plant.

The term “transgenic organism” does not cover native nucleotide codingsequences in their natural environment when they are under the controlof their native promoter which is also in its natural environment.

Therefore, the transgenic organism of the present invention includes anorganism comprising any one of, or combinations of, a nucleotidesequence coding for a polypeptide having the specific properties asdefined herein, constructs as defined herein, vectors as defined herein,plasmids as defined herein, cells as defined herein, or the productsthereof. For example the transgenic organism can also comprise anucleotide sequence coding for a polypeptide having the specificproperties as defined herein under the control of a promoter notassociated with a sequence encoding a hydrophobin or a membrane protein.

Transformation of Host Cells/Organism

The host organism can be a prokaryotic or a eukaryotic organism.

Examples of suitable prokaryotic hosts include bacteria such as E. coliand Bacillus licheniformis.

Teachings on the transformation of prokaryotic hosts are well documentedin the art, for example see Sambrook et al. (Molecular Cloning: ALaboratory Manual, 2nd edition, 1989, Cold Spring Harbor LaboratoryPress). If a prokaryotic host is used then the nucleotide sequence mayneed to be suitably modified before transformation, e.g., such as byremoval of introns.

In another embodiment the transgenic organism can be a yeast.

Filamentous fungi cells may be transformed using various methods knownin the art—such as a process involving protoplast formation andtransformation of the protoplasts followed by regeneration of the cellwall in a manner known. The use of Aspergillus as a host microorganismis described in EP 0 238 023. In one embodiment, T. reesei is the hostorganism.

Another host organism can be a plant. A review of the general techniquesused for transforming plants may be found in articles by Potrykus (AnnuRev Plant Physiol Plant Mol Biol (1991) 42:205-225) and Christou(Agro-Food-Industry Hi-Tech Mar./Apr. 1994 17-27). Further teachings onplant transformation may be found in EP-A-0449375.

General teachings on the transformation of fungi, yeasts and plants arepresented in following sections.

a. Transformed Fungus

A host organism may be a fungus—such as a filamentous fungus. Examplesof suitable such hosts include any member belonging to the generaFusarium, Thermomyces, Acremonium, Aspergillus, Penicillium, Mucor,Neurospora, Trichoderma and the like. In one embodiment, Trichoderma isthe host organism, preferably T. reesei.

Teachings on transforming filamentous fungi are reviewed in U.S. Pat.No. 5,741,665 which states that standard techniques for transformationof filamentous fungi and culturing the fungi are well known in the art.An extensive review of techniques as applied to N. crassa is found, forexample in Davis and de Serres, Methods Enzymol (1971) 17A: 79-143.

Further teachings on transforming filamentous fungi are reviewed in U.S.Pat. No. 5,674,707.

In one aspect, the host organism can be of the genus Aspergillus, suchas Aspergillus niger.

A transgenic Aspergillus according to the present invention can also beprepared by following, for example, the teachings of Turner G. 1994(Vectors for genetic manipulation. In: Martinelli S. D., Kinghorn J. R.(Editors) Aspergillus: 50 years on. Progress in industrial microbiologyvol 29. Elsevier Amsterdam 1994. pp. 641-666).

Gene expression in filamentous fungi has been reviewed in Punt et al.Trends Biotechnol. (2002); 20(5):200-6, Archer & Peberdy Crit. Rev.Biotechnol. (1997)/7:273-306.

b. Transformed Yeast

In another embodiment, the transgenic organism can be a yeast.

A review of the principles of heterologous gene expression in yeast areprovided in, for example, Methods Mol Biol (1995), 49:341-54, and CurrOpin Biotechnol (1997); 8:554-60.

In this regard, yeast—such as the species Saccharomyces cerevisi orPichia pastoris or Hansenula polymorpha (see FEMS Microbiol Rev (200024:45-66), may be used as a vehicle for heterologous gene expression.

A review of the principles of heterologous gene expression inSaccharomyces cerevisiae and secretion of gene products is given by EHinchcliffe E Kenny (1993, “Yeast as a vehicle for the expression ofheterologous genes”, Yeasts, Vol 5, Anthony H Rose and J. StuartHarrison, eds, 2nd edition, Academic Press Ltd.).

For the transformation of yeast, several transformation protocols havebeen developed. For example, a transgenic Saccharomyces according to thepresent invention can be prepared by following the teachings of Hinnenet al., (1978, Proceedings of the National Academy of Sciences of theUSA 75, 1929); Beggs, J D (1978, Nature, London, 275, 104); and Ito, Het al. (1983, J Bacteriology 153, 163-168).

The transformed yeast cells may be selected using various selectivemarkers—such as auxotrophic markers dominant antibiotic resistancemarkers.

A suitable yeast host organism can be selected from thebiotechnologically relevant yeasts species such as, but not limited to,yeast species selected from Pichia spp., Hansenula spp., Kluyveromyces,Yarrowinia spp., Saccharomyces spp., including S. cerevisiae, orSchizosaccharomyce spp., including Schizosaccharomyce pombe.

A strain of the methylotrophic yeast species Pichia pastoris may be usedas the host organism.

In one embodiment, the host organism may be a Hansenula species, such asH. polymorpha (as described in WO 01/39544).

c. Transformed Plants/Plant Cells

A host organism suitable for the present invention may be a plant. Areview of the general techniques may be found in articles by Potrykus(Annu Rev Plant Physiol Plant Mol Biol (1991) 42:205-225) and Christou(Agro-Food-Industry Hi-Tech Mar./Apr. 1994 17-27), or in WO 01/16308.The transgenic plant may produce enhanced levels of phytosterol estersand phytostanol esters, for example.

Culturing and Production

Host cells transformed with the nucleotide sequence of the presentinvention may be cultured under conditions conducive to the productionof the encoded polypeptide and which facilitate recovery of thepolypeptide from the cells and/or culture medium.

The medium used to cultivate the cells may be any conventional mediumsuitable for growing the host cell in questions and obtaining expressionof the enzyme.

The protein produced by a recombinant cell may be displayed on thesurface of the cell.

The polypeptide may be secreted from the host cells and may convenientlybe recovered from the culture medium using well-known procedures.

Secretion

Often, it is desirable for the polypeptide to be secreted from theexpression host into the culture medium from where the enzyme may bemore easily recovered. According to the present invention, the secretionleader sequence may be selected on the basis of the desired expressionhost. Hybrid signal sequences may also be used with the context of thepresent invention.

Typical examples of secretion leader sequences not associated with anucleotide sequence encoding a lipid acyltransferase in nature are thoseoriginating from the fungal amyloglucosidase (AG) gene (glaA—both 18 and24 amino acid versions e.g., from Aspergillus), the a-factor gene(yeasts e.g., Saccharomyces, Kluyveromyces and Hansenula) or thea-amylase gene (Bacillus).

Detection

A variety of protocols for detecting and measuring the expression of theamino acid sequence are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescentactivated cell sorting (FACS).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and can be used in various nucleic and amino acidassays.

A number of companies such as Pharmacia Biotech (Piscataway, N.J., USA),Promega (Madison, Wis., USA), and US Biochemical Corp (Cleveland, Ohio,USA) supply commercial kits and protocols for these procedures.

Suitable reporter molecules or labels include those radionuclides,enzymes, fluorescent, chemiluminescent, or chromogenic agents as well assubstrates, cofactors, inhibitors, magnetic particles and the like.Patents teaching the use of such labels include U.S. Pat. No. 3,817,837;3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241.

Also, recombinant immunoglobulins may be produced as shown in U.S. Pat.No. 4,816,567.

Fusion Proteins

A polypeptide for use in the present invention may be produced as afusion protein, for example to aid in extraction and purificationthereof. Examples of fusion protein partners includeglutathione-S-transferase (GST), 6×His, GAL4 (DNA binding and/ortranscriptional activation domains) and β-galactosidase. It may also beconvenient to include a proteolytic cleavage site between the fusionprotein partner and the protein sequence of interest to allow removal offusion protein sequences. Preferably the fusion protein will not hinderthe activity of the protein sequence.

Gene fusion expression systems in E. coli have been reviewed in Curr.Opin. Biotechnol. (1995) 6:501-6.

The amino acid sequence of a polypeptide having the specific propertiesas defined herein may be ligated to a non-native sequence to encode afusion protein. For example, for screening of peptide libraries foragents capable of affecting the substance activity, it may be useful toencode a chimeric substance expressing a non-native epitope that isrecognized by a commercially available antibody.

Additional Proteins

The biomimetic membranes described herein may also be used inconjunction with one or more additional proteins of interest (POIs) ornucleotide sequences of interest (NOIs).

Non-limiting examples of POIs include: proteins or enzymes involved instarch metabolism, proteins or enzymes involved in glycogen metabolism,acetyl esterases, aminopeptidases, amylases, arabinases,arabinofuranosidases, carboxypeptidases, catalases, cellulases,chitinases, chymosin, cutinase, deoxyribonucleases, epimerases,esterases, αgalactosidases, galactosidases, αglucanases, glucan lysases,endo-β-glucanases, glucoamylases, glucose oxidases, αglucosidases,β-glucosidases, glucuronidases, hemicellulases, hexose oxidases,hydrolases, invertases, isomerases, laccases, lipases, lyases,mannosidases, oxidases, oxidoreductases, pectate lyases, pectin acetylesterases, pectin depolymerases, pectin methyl esterases, pectinolyticenzymes, peroxidases, phenoloxidases, phytases, polygalacturonases,proteases, rhamno-galacturonases, ribonucleases, thaumatin,transferases, transport proteins, transglutaminases, xylanases, hexoseoxidase (D-hexose: O2-oxidoreductase, EC 1.1.3.5) or combinationsthereof. The NOI may even be an antisense sequence for any of thosesequences.

The POI may even be a fusion protein, for example to aid in extractionand purification.

The POI may even be fused to a secretion sequence.

EXAMPLES

The present disclosure is described in further detail in the followingexamples, which are not in any way intended to limit the scope of thedisclosure as claimed. The attached figures are meant to be consideredas integral parts of the specification and description of thedisclosure. The following examples are offered to illustrate, but not tolimit the claimed disclosure.

Example 1

Materials and Methods

Sodium dodecyl sulfate (SDS),1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),L-α-Phosphatidylcholine (SoyPC), Tris-Base buffer (Sigma-Aldrich), andHydrophobin (HFBII)

Micelles Formation:

SDS micelles were formed in 5 mM Tris buffer, 150 mM NaCl pH 7. Anappropriate amount of SDS was dissolved in the buffer for getting afinal concentration of 0.035M. The samples were shaken in a Vortex for 1min. to ensure a final homogeneous solution.

Micelles-Protein Samples:

SDS micelles-HFBII mixtures were formed in 5 mM Tris buffer, 150 mMNaCl, pH 7, at a protein-detergent molar ratio of 1:07 (molar ratio) Theoptimal HFBII:SDS ratio was selected according to the data published byZhang et al. (Zhang et al. 2011 Adsorption Behavior of Hydrophobin andHydrophobin/Suifactant Mixtures at the Solid-Solution Inteiface.Langmuir 27:10464-74; Zhang et al. 2011. Self-Assembly of Hydrophobinand Hydrophobin/Suifactant Mixtures in Aqueous Solution. Langmuir27:10514-22; Zhang et al. 2011. Adsorption Behavior of Hydrophobin andHydrophobin/Suifactant Mixtures at the Air-Water Inteiface. Langmuir 27:113616-23). When SDS-HFBII are mixed in solution although soft manualmixing was applied to avoid the development of big amounts of foam, themixture evolved into a gelatinous mixture which avoids any furthermanipulation of the sample. Alternatively the mixtures where performedin situ in the surface tension equipment.

In situ procedure: The different components of the mixture were added inconsecutives steps to the air/water interface. First the correct volumeof SDS micelles and afterwards the expected HFBII volume for achievingthe expected 1:0,7 (molar ratio). HFBII addition step indicated witharrows in the FIG. 3.

Liposomes-Small Unilamellar Vesicles (SUV) Formation:

DOPE:SoyPC 7:3 (mol ratio) vesicles were formed in 5 mM Tris buffer, 150mM NaCl, pH 7. Stock solutions of the phospholipids were prepared inchloroform:Methanol (2:1, v/v). An appropriate amount of the stocksolution was deposited in an assay tube. The organic volume wasevaporated under N2 flux and later over-night (o/n) in a desiccator forensuring the organic solvent removal. Afterwards the lipid film washydrated for 1 h at constant temperature (45° C.) with the appropriateamount of buffer for reaching the desired final concentration. Inaddition, occasional vortexing was applied to avoid material deposition.

SUV (<50 nm) was obtained using a Branson 250/450 tip sonifier at 360W/cm2 during three cycles of 30 s at intervals of 30 s to avoid sampleheating.

Liposomes-Protein Samples:

Lipid-HFBII samples were formed in 5 mM Tris buffer, 150 mM NaCl, pH 7.The same procedure explained above for the SUV formation was followed,with the exception that HFBII was added together with the buffer in thehydration process to get a final lipid concentration in the mixture of 1mg/mL. The final HFBII-Lipid molar ratio was 1:07 following the ratioused with the SDS micelles. The mixing, incubation and sonication stepswere the same.

Tensiometer K100:

Interfacial surface tension of the samples was registered vs. time.Samples volumes of 10 or 20 μL were deposited on the 35 mL buffersurface at a controlled temperature of 25° C.

Results and Discussion

FIG. 1 depicts a graph showing all the surface tension curves vs. time.For facilitating the comparison and the discussion the curves have beensplit in two different graphics. FIG. 3 depicts a graph in which datarelated to SDS and SDS/HFBII has been plotted. FIG. 4 depicts a graph inwhich data related to Liposomes and Liposomes/HFBII mixtures have beenplotted.

To our knowledge there is no exiting data on HFB-liposomes that can beused as a reference. However SDS-HFB has been already very wellcharacterized by Zhang et al. (Zhang et al. 2011 Adsorption Behavior ofHydrophobin and Hydrophobin/Suifactant Mixtures at the Solid-SolutionInteiface. Langmuir 27:10464-74; Zhang et al. 2011. Self-Assembly ofHydrophobin and Hydrophobin/Suifactant Mixtures in Aqueous Solution.Langmuir 27:10514-22; Zhang et al. 2011. Adsorption Behavior ofHydrophobin and Hydrophobin/Suifactant Mixtures at the Air-WaterInterface. Langmuir 27:113616-23). Adsorption Behavior of Hydrophobinand Hydrophobin/Surfactant Mixtures at the Solid-Solution Interface.Langmuir 27:10464-74; Zhang et al (2011). Self-Assembly of Hydrophobinand Hydrophobin/Surfactant Mixtures in Aqueous Solution. Langmuir27:10514-22; Zhang et al (2011). Therefore, SDS-HFB mixtures have beenadded as reference data. According to those references the HFBII-SDSmolar ratio of 1:0.7 is the optimal one for obtaining the maximumadsorption of the mixture into an air-water interface. Otherwise itwould be a competition between SDS and HFBII and the interface would bemainly occupied by HFBII.

The minimum volume allowed (35 mL) in the Tensiomiter K100 cell (FIG. 2)have been used in these experiments. A spreading set up was followedinstead of following the adsorption set up. In the adsorption set up thesample would be a) injected from the bottom of the Tensiometer cell orb) premixed with the buffer before starting the surface tensionregistration. In the spreading setup the sample is dropped on the top ofthe buffer. The surface tension of the buffer is registered from the t=0and it is confirmed that tensioactive molecules are not present in thesolution. The sample is then dropped on the surface. If the mixture istensioactive the surface tension will drop. In the spreading set up itis possible to work with very small volumes, μL, of samples notnecessarily very concentrated as soon as they are containing tensoactivemolecules.

FIG. 3 shows the surface tension curves of SDS, HFBII and SDS/HFBIIsamples. 10 μL of the SDS stock solution was dropped on the surface. Dueto the deposition the surface tension is dropping intensively, but oncethe SDS molecules are stably spread on air/water interface the surfacethe surface tension values are very close to the pure buffer. Thisindicates that SDS molecules at that concentration are not showing ahigh tensioactivity.

10 or 20 μL from the HFBII stock solution were deposited on the buffersurface. It seems that HFBII spreading is happening in two welldifferentiated phases. It is probable that the first phase arereflecting the HFBII reorganization on the surface and once the moleculeare covering the full surface then the maximum tensoactive value isreached for the HFBII layer formed on the surface.

Due to the limitation of working with premixed SDS/HFBII samples themolecules were added in two different steps. 10 μL from the SDS stocksolution was added to the samples and immediately followed by theaddition of 10 μL from the HFBII stock solution. The curves registeredfrom these samples are having a complex profile. The first intense dropon the surface tension is due to the SDS addition and this is slowed bythe addition of the HFBII. The spreading of the HFBII is showing adifferent spreading kinetic than when is not facing SDS in the surface.Duplicates of these samples are not completely equal and the mostprobable reason is small differences in the way HFBII facing the SDSsurface. But the HFBII presence reverts without any doubt tensioactivitylost shown by the pure SDS samples. The mixture is reaching the sametensoactive values than HFBII alone.

FIG. 4 shows results with liposomes and HFBII-liposomes samples. Thephospholipid liposomes have shown the expected low surface tensionprofile. In presence of the HFBII the surface tension dropped to typicalvalues observed on tensioactive phospholipid-protein membranes mixtures.Interestingly, if the sample is tested right after preparation, thesample showed the expected phospholipid-protein mixture profile.However, if the same sample is tested after being kept at roomtemperature without any agitation during 4 h, the curve showed abiphasic behavior very similar to the one observed on HFBII alone. Thissuggests that HFBII is leading the sample profile. However, if the samesample is sonicated in a water bath for one minute, the profile is backto the one observed on the freshly prepared sample showing the samesurface tension stable profile. This would indicate that whilein-solution interactions between HFBII and the phopholipids is notcompletely stable (and that is changing with the passing of time), thisis a reversible process that is not limiting the stability properties ofHFBII on phospholipids membranes, i.e. the resulting interfacialinteraction remains highly stable.

These results indicate that HFBII can stabilize phospholipids membranes.

Example 2

AqpZ-HFBII Mixtures

Materials:

AqpZ: 20 mg/mL stock solution containing 20 mM Tris, 300 mM NaCl, 300 mMImidazole, 10% glycerol, 0.3% Surfactant, pH 8.0

HFBII: 8.5% stock solution containing 0.13% potassium sorbate, pH 3.5

Procedure:

4 solutions containing AqpZ and HFBII mixtures were prepared withdifferent AqpZ and HFBII concentrations in 10 mM PBS buffer (pH=7.6)(Samples 1-4). The samples were dialyzed (cutoff=1 kDa) against 10 mMPBS buffer for 72 hours.

The surfactant was removed via dialysis which exposes the hydrophibcsurfaces of AqpZ to be accessed by HFBII.

For comparison, 4 solutions containing only AqpZ or HFBII were alsoprepared (Control 1-4) and subject to the same dialysis process.

AqpZ HFB 1000 ppm 100 ppm 0 ppm 1000 ppm  Sample 1 Sample 2 Control 3100 ppm Sample 3 Sample 4 Control 4  0 ppm Control 1 Control 2

Characterization:

The mixtures were examined with dynamic light scattering (DLS) using aMelvern nano Zetasizer (Malvern Instruments Ltd, Worcestershire, UK). Adisposable cuvette was first cleaned with nitrogen gas to remove dust.Then 400 μl of the sample was placed in the cuvette. The cuvette wasinserted into the Zetasizer. DTS (Nano) software (Malvern InstrumentsLtd) was used to determine the particle size.

Results & Discussions:

The objective of this experiment is to examine the interaction betweenAqpZ and HFBII proteins. Such interaction may be utilized to stabilizeAqpZ dispersion, immobilize AqpZ on substrates and in addition createcomplex functional systems.

Herein we investigated 4 mixtures at different AqpZ and HFBIIconcentrations (Samples 1-4) using DLS and results are summarized belowand in FIGS. 15A and 15B.

All samples after 72 hour dialysis remained stable and homogeneous. Thecontrol sample 1 with 1000 ppm AqpZ showed a broad particle sizedistribution centered at 9 nm. Especially, the long tail at the leftside of the curve suggests the presence of large aggregated structures.In comparison, samples with the same AqpZ concentration and additional1000 ppm or 1000 ppm HFBII (Samples 1 and 3) showed the same meanparticle size but narrower size distribution, indicating HFBII may haveassisted the dispersion of AqpZ in solution. It is worth mentioning thatHFBII also forms particles of 4˜10 nm (data not shown).

At 100 ppm AqpZ concentration, the effect of HFBII to help disperse AqpZis more prominent. While 100 ppm AqpZ formed aggregates >1 μm, steaddecrease in average particle size was clearly observed with the additionof 100 ppm and 1000 ppm HFBII. (Samples 2 and 4) Especially atHFBII/AqpZ ratio of 10 (Sample 4), two orders of magnitude increase instructural size was registered. Those structures cannot be attributed toHFBII only which has an average particle size of 4˜10 nm (data notshown). Therefore, it is possible that AqpZ/HFBII complexes have beenformed under those conditions.

The reason pure AqpZ dispersion is more stable at 1000 ppm than 100 ppmis probably because of the residue surfactant.

These results suggest the presence of interaction between AqpZ and HFBIIprotein, perhaps in multiple modes as a function of theirconcentrations. Such interaction can be utilized to disperse andstabilize AqpZ in solution.

Example 3

Surface Modification of Block Copolymer Membranes by HFBII

Materials: PMOXA₁₅-PDMS₆₇-PMOXA₁₅ was acquired from Sigma-Aldrich.HFBII: 8.5% stock solution containing 0.13% potassium sorbate, pH 3.5.

Procedure:

PMOXA₁₅-PDMS₆₇-PMOXA₁₅ was dissolved in chloroform with rigorousagitation and left still at room temperature to reach homogeneity. Blockcopolymer membranes were prepared by casting ˜0.2 mL chloroform solutiononto triple DI water washed glass slide (1 in.×3 in.) and dried in airfor 72 hrs. The resulting membrane was further dried in a vacuum oven at0.3 mbar at room temperature for 60 min to remove remaining chloroform.For HFB surface modification, the glass slide with polymer membrane wassoaked in 1000 ppm HFB solution for 30 min, followed by washing with DIwater and dried in vacuum oven at 0.3 mbar for another 60 min. Forcomparison, another piece of the same membrane sample was soaked inwater for the same duration and subject to the same drying procedure.

Characterization:

A ramé-hart standard Goniometer (ramé-hart Instrument Co., Netcong,N.J., United States) was used for WCA determinations. The substrate wasplaced in the Goniometer and leveled. A water drop was placed onto thesurface using a syringe. The software DROPimage Advanced (ramé-hartInstrument Co.) was used to measure the water contact angles, bydefining the bottom of the droplet and profiling the drop. The left andright contact angles of the drop were averaged. This method was repeatedin different locations on the substrate surface until the contact anglesof five water drops were determined and averaged.

Results & Discussions:

FIG. 16 summarizes the water contact angle results for glass substrate,BCP coated glass, BCP coated glass after HFB modification, and a controlsample of BCP coated glass soaked in water. While the BCP film hassignificantly lower water contact angle (18±4°) compared to glasssubstrate (57±6°), treatment with HFB further reduced the water contactangle to 8±3°. In comparison, BCP film treated with water showedslightly increased water contact angle (26±3°), indicating the BCP filmmay be partially dissolved by water due to insufficient cross-linking.

The results suggest the presence of HFB coating on theMOXA₂₀₀₀-DMS₄₀₀₀-MOXA₂₀₀₀ film and it may help stabilize block copolymerfilm/membrane, either in planar or vesicle forms. (Reference: S-layerprotein stabilization of block copolymer membrane (Desalination bybiomimetic aquaporin membranes: Review of status and prospects.Desalination 308:34-40; Tang et al (2013))

Example 4

AQPZ/HFBII Proteoliposomes Preparation

AqpZ Expression and Purification

Both wild type AqpZ and an inactive mutantAqpZR189A (i.e., a mutant withlow water permeability) can be expressed and used in the presentexample. The inactive mutant can be used as a negative control to theAqpZ wild type. Genomic DNA from E. coli DH5a can be employed as asource for amplifying the AqpZ gene, using gene specific primers withthe addition of 6-Histag sequence at the N-terminus. The amplified AqpZis digested with the enzyme NdeI and BamH I and then ligated to thesimilarly digested pEt3a vector DNA. The positive clones are verified byPCR-screening. Then the authenticity of the constructs is confirmed byDNA sequencing (1st-base). ForAqpZR189A, the arginine residue atposition 189 is replaced with alanine to the pET3a/AqpZ using theQuikchange™ site-directed mutagenesis (SDM) kit. The mutagenesisconstructs is confirmed by DNA sequencing (1st-base).

Expression and purification of both AqpZ wild type and AqpZ R189A isperformed according to methods known in the art. The purified ApqZ wildtype and AqpZR189A are stocked in buffer (20 mM TrispH8.0, 300 mMNaCl,300 mM Imidazole, 2 mM b-mercaptoethanol, 10% glycerol, containing 30mMn-octyl b-DGlucopyranoside) and kept frozen at 80° C. Proteinconcentration is determined by UV absorbance at 280 nm using a Nanodrop1000 spectrophotometer (ThermoScientific, Massachusetts, USA, AqpZextinction coefficient=35,090 M⁻¹cm⁻¹, molecular weight=24,524 g/mol).

AqpZ and HFBII Incorporation

Lipid vesicles (i.e., liposomes) are prepared by the film rehydrationmethod. Briefly, a thin lipid film is formed by drying a 10 mg DOPClipid dissolved in 0.5 ml chloroform solution under nitrogen headspace.The DOPC film is kept in a vacuum desiccators for at least 2 h, before a1 ml phosphate buffered saline (PBS) buffer solution (pH7.4) is added torehydrate the film followed by 3 cycles of freeze-thaw treatment. Theresulting solution, containing unilamellar liposomes, is extrudedthrough a 200 nm pore size polycarbonate filter 21 times (Avestinextruder, Avestin, Canada) to obtain liposomes with uniform sizedistribution.

Proteoliposomes are prepared using the dialysis method by incorporatingAqpZs (both wild type and the inactive mutant) and HFBII at differentconcentrations (0.01-20 mM) into the DOPC liposomes. Briefly, a 10 mg/mlDOPC liposome suspension containing 1% detergent OG and an AqpZ/HFBIIsolution are mixed at a protein to lipid molar ratio of 1:200 for AqpZ,and different concentrations between a 0.1-20 mM is then removed fromthe proteoliposome solution by dialyzing against a PBS buffer solutionfor 3 days.

Membrane Substrate Preparation

Microporous membrane substrates are formed by the phase inversionmethod. Briefly, the polymer dope (16 wt % PS, 77 wt % NMP, 5 wt % PEG,and 2 wt % LiC1) is stirred at 70° C., degassed, and cooled down to roomtemperature (23° C.). A 15 ml sample of dope is cast on a clean glassplate with a thickness of 200 mm using an Elcometer 4340 Motorized FilmApplicator (Elcometer Asia Pte Ltd.). The glass plate with the nascentfilm is smoothly immersed into a coagulant bath (tap water at roomtemperature) to form the PS substrate.

Membrane Rejection Layer Preparation

The synthesis procedures for forming the membrane rejection layer isdepicted in Y. Zhao et al., (Synthesis of robust and high-performancebased biomimetic membranes by interfacial polymerization-membrane . . ., Journal of Membrane Science (2012),http://dx.doi.org/10.1016/j.memsci.2012.08.039), where Diaminobenzene(MPD) and Trimesoly Chloride (TMC) are used as monomers to form asalt-rejection polyamide layer via inter-facial polymerization whereasAqpZ/HFBII containing proteoliposomes are added to create preferentialwater paths in the rejection layer. The PS substrate is first soaked ina MPD aqueous solution containing 1% MPD, 0.1% SDS, and 10 mg/mlproteoliposomes (either AqpZ wild type/HFBII or AqpZ R189A/HFBII) for 10min. The soaked substrate is held horizontally at room temperature for30 min, and the excess aqueous solution is removed by compressednitrogen gas. The TMC solution (0.1% in n-Hexane) is then gently pouredon to the substrate surface to react with the residue MPD to form therejection layer. The resulting membranes are kept in flowing tap waterovernight to remove excess monomers and are stored in Milli-Q water.Additional membranes without the inclusion of proteoliposomes (i.e.,conventional TFC polyamide membranes) are also prepared for comparisonpurposes.

Liposome and Proteoliposome Characterization

The sizes of liposomes and proteoliposomes are determined using aNanoZetasizer (NanoZS, Malvern Instruments Limited, UK). Their waterpermeability is characterized by an SX20 Stopped-Flow Spectrometer(Applied Photophysics, UK). The fluorescence kinetic mode is chosen forthe stopped-flow test, with a light source of 500 nm wavelength. Rapidmixing of vesicle solution and draw solution (sucrose) is driven by 8atm pressurized nitrogen gas with a dead time of 500 ms. The vesiclevolume reduction as a result of water transport out of the vesiclesunder the osmolarity gradient is monitored, and used for waterpermeability (Pf) calculation:P _(f) =k/(S/V ₀ V _(w) Δosm)where S/V0 is the surface area to initial volume ratio of the vesicle,Vw is the partial molar volume of water (18 cm3/mol), and Δosm is thedifference in osmolarity between the intravesicular and extra vesicularaqueous solutions. The rate constant k is determined by curve fitting ofthe stopped-flow results with single order exponential. The k values arethe averages obtained from 5 to 10 independent stopped flowmeasurements.

Membrane Characterization

Microscopic Characterization.

Membranes are dried in a vacuum desiccators for 24 h before microscopiccharacterization.

Atomic force micrographs (AFM) are captured by a Park Systems XE-100 AFM(USA). Samples, sputter-coated with a thin film of gold, are also imagedby an FE-SEM (Field-emission scanning electron microscopy, JSM-7600F,JEOL, Japan).

Membrane Separation Properties.

Separation properties of the synthesized biomimetic membranes arecharacterized in a standard cross flow reverse osmosis set up. Briefly,a membrane coupon with an active surface area of 42 cm² is mounted inthe test cell (CF42 Membrane Cell, Sterlitech). The feed solution (10 mMNaCl at 2070.5 1 C) is pumped at a constant cross flow velocity of 20cm/s. A diamond-shaped feed spacer is used to minimize concentrationpolarization. Membranes are compacted for 2 h at the desired testingpressure before sample collection for water flux and rejectionmeasurements. Membrane permeate flux (Jv) is determined using thegravimetric method, and NaCl rejection (R) is obtained based onconductivity measurements (Ultrameter II, Myron L Company, Carlsbad,Calif.) of the permeate and feed water. The water permeability (A) andNaCl permeability (B) are calculated by the following equations,respectivelyA=J _(V)/(ΔP−Δπ)B=(1/R−1)J _(V)where ΔP is the applied pressure and Δπ is the osmotic pressuredifference across the membrane.

Example 5

Liposome and Proteoliposome Preparation

Lipid vesicles are prepared by the film rehydration method. Briefly, A10 mg lipid dissolved in 0.5 ml chloroform is dried under nitrogen gasto form a thin lipid film. In some experiments, a predetermined amountof cholesterol can be is kept in a vacuum desiccator for at least 2hours. A 1 ml phosphate buffered saline (PBS) buffer solution (pH 7.4)is used to rehydrate the lipid film, followed by 3 cycles of freeze thawtreatment. The resulting solution contains unilamellar lipid vesicleswith wide size distribution. Liposomes with uniform size are obtained byextruding the solution through a 200 nm pore size polycarbonate filterfor 21 times using an extruder. Proteoliposomes are prepared byincorporating AqpZ and HFBII into liposome by dialysis. Briefly, anAqpZ/HFBII solution is mixed with a second solution containing 10 mg/mllipid vesicles and 1% detergent OG at a lipid to protein ratio of 200:1,followed by incubating at room temperature for 1 hr. Dialysis tubing(Spectrum laboratories, USA, with MWCO 12-14 KDa) is used to remove OGfrom the proteoliposome solution by dialyzing it against a PBS buffersolution at pH 7.4 for 3 days. During this period, the dialysis PBSbuffer solution is changed once every day. After the 3-day dialysis,AqpZ (wild type or R189A) and HFBII should be successfully reconstitutedinto lipid vesicles.

Preparation of a Biomimetic Membrane Having IncorporatedLipid-AqpZ/HFBII Vesicles

A commercial UF membrane (MWCO, 50,000 Dalton) is used as substrate, 50ml aqueous amine solution 1.5 wt. % MPD containing 0.08 mg/gDOPC-AqpZ/HFBII vesicles is spread onto the surface of the UF membranesubstrate, and kept the substrate wet with aqueous solution for 15 min.After that, the aqueous amine solution is removed from the surface andthe substrate is standed vertically in the air for 10 min, followed byblowing the surface to remove any possible aggregated vesicles onto thesurface with compressed nitrogen gas at 2 bar for 1 min. Then thesubstrate continues to stand vertically to dry for another 20 min.Subsequently, a 0.1 w/v % TMC solution is poured on the skin layer ofthe saturated substrate and reacted for 1 min. The resultant membrane isstored in Milli-Q water till to use. The resulting membrane is fixed ina testing cell. Feed solution (500 ppm NaCI) is pumped at 200 psi fromthe feed tank, flowed against the active layer of membrane and is thenreturned to the tank. Permeate is collected and the weight is measuredand concentration of solute is determined by conductivity measurement inorder to calculate flux and rejection.

Example 6

Polymersome Preparation

PMOXA₁₅-PDMS₆₇-PMOXA-₁₅ is dissolved in chloroform with rigorousagitation and left still at room temperature to the homogeneous mixingtogether. The concentration in the solution should be from 1.0 to 20.0w/v. % (8-12 w/v.%). Then, the chloroform is evaporated under nitrogenpurge in a rotary vapor evaporator. The copolymer is further dried in avacuum oven at 0.3 mbar at room temperature overnight to remove thetrace amount of remaining solvent. After that, 1 ml of PBS solution isadded to the dried block copolymer and the mixture is again subject torigorous agitation for a pre-determined duration. The diameter ofpolymersome vesicle is obtained by TEM image. The diameter of thepolymersome vesicle could vary from 200 nm to 350 nm

Preparation of Polymersome-AqpZ/HFBII Vesicles

The process of preparation of polymersome-AqpZ/HFBII vesicles is thesame as lipid vesicles as described in the Examples above, but thepolymer to AqpZ/HFBII ratio is changed to 20:1-500:1. Vesicles usingdifferent ratios within this range are spread into UF membranes aretested as described below.

Preparation of a Composite Membrane Having Incorporated CopolymerVesicles

The composition of reactive monomer solution in two phase and theprocess of interfacial polymerization are similar to Example 3, except,0.08 mg/g (PMOXA₁₅-PDMS₆₇-PMOXA-₁₅) polymersome vesicles with or withoutAqpZ/HFBII are dissolved in the aqueous amine solution. Reverse osmosistesting is done as in Example 3.

Example 7

Encapsulation of Proteins and Reconstitution of Channel Proteins inPMOXA-PDMS-PMOXA Triblock Copolymer Vesicles

For the reconstitution experiments, the well-characterized bacterialporin OmpF can be used. A porin stock solution (13.3 mg mL.sup.-1 in 1wt % octyl-polyoxyethylene, 100 mM NaCl, and 2.5 mM Hepes, pH 7.4) ismixed with different concentration of HFBII stock solution (8.5% stocksolution containing 0.13% potassium sorbate, pH 3.5) and 17 wt %solution ofPoly(2-methyloxazoline)-block-poly(dimethylsiloxane)-block-poly(2-methyloxazoline)(PMOXA-PDMS-PMOXA) triblock copolymer in ethanol to a final molar ratioof 1:1000 (porin:polymer). For encapsulation of the enzymebeta-lactamase in the interior of the vesicle, the solution is slowlyadded to an aqueous beta-lactamase solution (0.024 mg/ml in 10 mM Hepes,100 mM NaCl, pH 7.4) to a final copolymer concentration of 1 wt %. Theresulting dispersion is extruded through Nucleopore filters, pore width200 nm, which should result in a monodisperse collection of vesicleshaving an average diameter of 250 nm³.

The vesicles can be polymerized by irradiating the vesicle dispersionfor 2 minutes with uv light.

10 μl of a 1 mM ampicillin solution (in 10 mM Hepes, 100 mM NaCl, pH7.4) is added to the nanocapsule dispersion and the mixture is incubatedfor 30 minutes. Starch iodine reagent is prepared by mixing 5 ml of a 8mM iodine, 320 mM potassium iodide solution with 20 ml 1M sodiumwolframate in 2 M acetic acid and then adding 5 ml of 2 wt % solublestarch which had been dissolved in 1 M acetic acid by boiling gently for3 minutes. Subsequently, 0.5 ml of the starch-iodine reagent is added tothe vesicle mixture and the absorbance of the starch-iodine complex wasat 623 nm is measured as a function of time.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

The invention claimed is:
 1. A method of making a stabilized biomimetic membrane comprising a membrane protein wherein the stabilized membrane is formed after adding a Class II hydrophobin to a biomimetic membrane forming solution, wherein the Class II hydrophobin is added at a concentration of 0.1 μM-50 mM.
 2. The method of claim 1, wherein the Class II hydrophobin has the general formula: (Y1)n-B1-(X1)a-B2-B3-(X3)c-B4-(X4)d-B5-(X5)e-B6-B7-(X7)g-B8 (Y2)m wherein: m and n are independently 0 to 200; B1, B2, B3, B4, B5, B6, B7 and B8 are each independently amino acids selected from Cys, Leu, Ala, Ser, Thr, Met or Gly, at least 6 of the residues B1 through B8 being Cys; a is 6 to 12; c is 8 to 16; d is 2 to 20; e is 4 to 12; and, g is 5 to
 15. 3. The method according to claim 2, wherein all 8 of the residues B1 through B8 are Cys.
 4. The method of claim 1, wherein said membrane protein is an aquaporin.
 5. The method of claim 4, wherein said aquaporin is AQPZ.
 6. The method of claim 4, wherein the aquaporin is of plant origin.
 7. The method according to claim 4, wherein the aquaporin is selected from the group consisting of a Tonoplast Intrinsic Protein, a Plasma Membrane Intrinsic Protein, and a Nodulin-26 like Intrinsic Protein aquaporin, and mixtures and hybrids thereof.
 8. The method according to claim 4, wherein the aquaporin is an aquaglyceroporin (GLpF).
 9. The method according to claim 8, wherein said GLpF is selected from the group consisting of a GLPA channel, a GLPB1 channel, a GLPB2 channel, a GLPB3 channel, and a GLPY2 channel, and mixtures and hybrids thereof.
 10. The method according to claim 1, wherein the Class II hydrophobin has a hydrophobin core sequence of between 40 and 120 amino acids.
 11. The method of claim 10, wherein said Class II hydrophobin is selected from the group consisting of HFBII (SEQ ID NO: 2) and HFBI (SEQ ID NO: 4), or wherein said Class II hydrophobin has a hydrophobin core that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or at least 99% sequence identity to the hydrophobin core of SEQ ID NO:
 2. 12. The method of claim 10, wherein said Class II hydrophobin is “HFBII” (SEQ ID NO: 2), or a protein having a hydrophobin core that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or at least 99% sequence identity to the hydrophobin core of SEQ ID NO:
 2. 13. The method according to claim 1, wherein the Class II hydrophobin is a Class II hydrophobin fusion protein.
 14. The method according to claim 1, wherein the Class II hydrophobin is obtained or obtainable from a filamentous fungus.
 15. The method according to claim 14, wherein the Class II hydrophobin is obtained or obtainable from a fungus of genus selected from the group consisting of Cladosporium, Ophistoma, Cryphonectria, Trichoderma, Gibberella, Neurospora, Maganaporthe, Hypocrea, Xanthoria, Emericella, Aspergillus, Paracoccioides, Metarhizium, Pleurotus, Coprinus, Dicotyonema, Flammulina, Schizophyllum, Agaricus, Pisolithus, Tricholoma, Pholioka, Talaromyces and Agrocybe.
 16. The method according to claim 1, wherein the Class II hydrophobin is added by being generated in situ in the biomimetic membrane forming solution.
 17. The method according to claim 1, wherein the Class II hydrophobin causes at least 1.2 fold increase in stability of the biomimetic membrane compared to the stability in the absence of said Class II hydrophobin.
 18. The method according to claim 1, wherein said Class II hydrophobin provides a decrease in an equilibrium surface tension at said biomimetic membrane below 50 mN/m.
 19. The method according to claim 1, wherein said Class II hydrophobin causes the surface shear elasticity at said biomimetic membrane to increase to 0.3-0.6 N/m or higher.
 20. The method according to claim 1, wherein the Class II hydrophobin has the general formula: (Y1)n-B1-(X1)a-B2-B3-(X3)c-B4-(X4)d-B5-(X5)e-B6-B7-(X7)g-B8-(Y2)m wherein: m and n are independently 0 to 10; B1, B2, B3, B4, B5, B6, B7 and B8 are each independently amino acids selected from Cys, Leu or Ser, at least 7 of the residues B1 through B8being Cys; a is 7 to 11; c is 11; d is 4 to 18; e is 6 to 10; and g is 7 to
 10. 